Diuretic agents

Diuretic Agents Edward J.

Cragoe, Jr.,

EDITOR

Merck Sharp and Dohme Research Laboratories

Based on a symposium sponsored by the A C S Division of Medicinal Chemistry at the 174th Meeting of the American Chemical Society, Chicago, Illinois, August 29,

ACS

SYMPOSIUM

AMERICAN

1977.

SERIES

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1978

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

83

Library of CongressCIPData Diuretic agents. (ACS symposium series; 83 ISSN 0097-6156) Includes bibliographies and index. 1. Diuretics and diuresis—Congresses. 2. Chemistry, Pharmaceutical—Congresses. I. Cragoe, Edward J. II. American Chemical Society. Division of Medicinal Chemistry. III. Series: American Chemical Society. ACS symposium series: 83. RS431.D58D58 ISBN 0-8412-0464-0

615'.761 ASCMC 8

78-23405 83 1-238 1978

Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED

STATES

OF

AMERICA

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series Robert F . G o u l d , Editor

Advisory Board Kenneth B. Bischoff

Nina I. McClelland

Donald G . Crosby

John B. Pfeiffer

Jeremiah P. Freeman

Joseph V . Rodricks

E. Desmond Goddard

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Robert A . Hofstader

Raymond B. Seymour

James P. Lodge

Roy L. Whistler

John L. Margrave

Aaron Wold

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

FOREWORD The ACS SYMPOSIU

a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

PREFACE Some scientists believe that man is a brain surrounded by cells of secondary significance while others are persuaded that the heart takes preeminence. O f course, neither is correct; man consists of two magnificent organs called kidneys which are imbedded in tissues of lesser importance. Seriously, the kidney provides a pervasive, homeostatic regulation of the entire internal environment of the body; more specifically, kidney function modulates electrolyte and fluid balances, the economy of nutrients and drugs, and the cause the kidney is involved either directly or indirectly with nearly every cellular process, the ability of this organ to function properly under conditions of stress, age, or disease becomes progressively more important and, ultimately often becomes the limiting factor in the survival of the body itself.

Since water and electrolyte are the major constituents of

terrestrial life, organs that control their transport are of vital importance. Since the beginning of modern medicine, the discovery and use of drugs which control renal function have been a major goal. Only a small portion of what may be considered to be the full potential of this field has been realized. Future achievements in this area are certain to come, albeit with many birth pangs. T h e first drugs developed for the control of renal function were the diuretics which also were among the first synthetic agents to be introduced into medicine. Although modern diuretic therapy is not yet three decades old, a series of prominent milestones mark its history, and this history is still being made. T h e outstanding and continuing record of the development of novel diuretic agents has greatly enhanced the progress of the basic science relating to kidney function. This fundamental knowledge has, in turn, permitted the discovery of even more unique diuretics. Most importantly, these drugs are as safe and sophisticated as any that are in our present medical armamentarium. Mercurial agents constituted the first chemical class of the synthetic diuretics, and these drugs possessed many commendable attributes, including a good electrolyte excretion profile, high potency, and uricosuric activity.

Within a few years, the carbonic anhydrase inhibitors were

discovered which provided a more practical therapy. T h e advent of the thiazides opened the door to a deluge of structurally and functionally related sulfonamide diuretics.

T h e loop diuretics, as represented vii

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

by

furosemide and ethacrynic acid, constituted the next major breakthrough. Finally, the antikaliuretic saluretics,

i.e., aldactone,

triamterene,

and

amiloride, entered the arena. During their short history in medicine, the demand for and the use of diuretics has increased dramatically. It is estimated that the worldwide use of diuretics involved ten billion patient days in 1977 at a cost of $765 million. If the use of diuretics increases at its present rate, it will double within the next five or six years.

Interestingly, the use of

diuretics for the treatment of hypertension is growing at a faster rate than that for edema. Another obvious trend is the shift away from the use of potassium-losing diuretics to that of the antikaliuretic diuretics, either alone or in combination. Research activity and the resultant major ^breakthroughs" in the diuretic field have occurred sporadically and, if the current signs are valid, we are in the mids Although a number of saf each have inherent deficiencies which impart to them a significant obsolescence liability.

T h e most prevalent problems include hypokalemia,

hyperglycemia, and hyperuricemia. In addition, most diuretics belong to a very small group of chemical or structural classes. As a result, they have similar mechanisms of action and side effects. T h e diuretics of the future must be structurally novel and mechanistically unique and, more importantly, they must obviate many of the side effects that are characteristic of the agents currently in use. The rapid advances that have been made in the sciences which are basic to the understanding of water and electrolyte transport have permitted a more sophisticated approach to the design of new diuretics.

It

also has allowed the early examination of new leads for their site and mechanism of action. Progress in the biochemistry of electrolyte transport has been noteworthy in recent years, especially in regard to the role of renal hormones. Likewise, advances in micropuncture and a variety of other techniques have provided tools which are useful for the routine evaluation and recognition of truly novel renal agents. It has been 15 years since deStevens' classic monograph (I)

on the

medicinal chemistry of diuretics appeared. Therefore, a symposium (2) was organized which was designed to review the progress that had occurred in the intervening years.

Since the symposium was limited to

only a one-half-day session, it was not possible to include reports from every institution where major discoveries had been made.

T h e interest

generated by the symposium suggested that the papers be published. In order to present a more complete picture of the innovative research in this field, both from the standpoint of the basic science and of the practical agents available at the clinical level, four more papers were added to those of the original symposium. viii

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Since the main purpose of this monograph was to describe the recent advances

i n the medicinal chemistry of diuretics, the emphasis

was

placed on the fundamental contributions of the medicinal chemists, i.e., drug design and structure-activity

relationships.

T h e authors of this

monograph and the institutions that they represent have made particu­ larly noteworthy contributions to renal research and many of them have been associated with a long history of outstanding work. O n the academic side, it seemed only appropriate to include the recent work on the prostaglandin and kallikrein-kinin systems.

T h e role

of these renal hormones is extremely complex and currently in a state of refinement, but it is of such importance that it demands consideration by all scientists involved in renal research. Examination of the history and current status of the diuretic field not only permits but encourages

extrapolation into the future.

Such

extrapolation suggests tha have novel structures, uniqu importantly, new types of renal agents will be discovered which will provide for the prophylaxis and treatment of renal disorders where no drug is currently available. Acknowledgments—Many

people have contributed to the planning

and programming of the diuretic symposium and to the writing of this book. T h e authors of each paper have been extremely cooperative and helpful, not only in providing their contribution, but in integrating their unit into the whole. M u c h editorial assistance and advice were provided by Ε . H . Blaine, M . G . Bock, S. J. deSolms, R. L . Smith, A . K . Willard, and O . W . Woltersdorf, Jr. I am indebted to Florence Berg for checking and editing the bibliographies of many of the manuscripts. I am most grateful to my secretary, C . F . Slobodzian, for the excep­ tional job she d i d i n typing and proofreading each draft of many of these manuscripts and of the final draft of most of the others. She also provided invaluable assistance in the

voluminous correspondence

required i n

organizing the symposium and writing the book. Literature

Cited—

1. deStevens, G . , "Medicinal Chemistry, a Series of Monographs, V o l . 1, Diuretics: Chemistry and Pharmacology," Academic, N e w York, 1963. 2. National

Meeting

of the

American

Chemical

Society, 174th, Sym­

posium on Diuretic Agents, Medicinal Chemistry papers 1-7, Merck Sharp and Dohme Research

1977.

EDWARD J. CRAGOE, JR.

Laboratories West Point, Pennsylvania August 21, 1978

ix

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Prostaglandins and Renal Function: Implications for the Activity of Diuretic Agents J. C. McGIFF and P. Y - K WONG Department of Pharmacology and Department of Medicine, University of Tennessee Center for the Health Sciences, Memphis, T N 38163

Prostaglandins ar act at or near their site demand as they are not stored (1). In the kidney, as in other tissues, prostaglandins serve primarily a defensive function, although they may contribute to the maintenance of renal function under physiological conditions. Furosemide, ethacrynic acid and bumetanide, the most potent of the diuretic agents, can cause a precipitous decline in renal function, particularly in the sodium depleted subject; a prostaglandin response evoked i n response to the "loop diuretic" may maintain renal function in the face of this challenge (2). The capacity of the kidney to respond to a stimulus which depresses renal function by increasing prosta­ glandin synthesis was f i r s t shown during administration of a vasoconstrictor agent such as angiotensin or norepinephrine (3). Release of prostaglandins coincided with restoration of renal blood flow and urine flow despite continued administration of either angiotensin II or norepinephrine. STRESS EVOKED RENAL PROSTAGLANDIN RESPONSE A prostaglandin mechanism seems important to the regulation of the renal circulation when the latter i s compromised by an acute insult or chronic disease. For example, activation of the renin-angiotensin system by either hemorrhage , laparotomy (5.) or a "loop diuretic" can increase synthesis of prostaglandins by the kidney; the concentration of PGE^ i n renal venous blood increased by as much as fifteen-fold during surgical stress and was closely correlated with the level of plasma renin activity (5). Thus, under acute stress the a c t i v i t i e s of the reninangiotensin and prostaglandin systems within the kidney appear to be coupled. The contribution of a prostaglandin mechanism to the support of the renal circulation in the acutely stressed dog may be uncovered by administration of indomethacin, an inhibitor of prostaglandin synthesis (5). A large reduction i n renal blood flow oceurred rapidly i n response to indomethacin, despite an 0-8412-0464-0/78/47-083-001$05Ό0/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

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attendant increase i n r e n a l p e r f u s i o n pressure. There was a simultaneous d e c l i n e i n r e n a l e f f l u x o f PGEp which was proport i o n a l to the r e d u c t i o n i n r e n a l blood flow. T h i s study demons t r a t e d that i n the animal subjected t o acute s t r e s s , the r e n a l c i r c u l a t i o n was supported by a major p r o s t a g l a n d i n component, withdrawal o f which r e s u l t e d i n decreased r e n a l blood flow, p a r t i c u l a r l y that f r a c t i o n to the inner cortex and medulla ( 7 ) . PROSTAGLANDIN RELATED EFFECTS ON RENAL BLOOD FLOW The pattern o f d i s t r i b u t i o n o f blood flow w i t h i n the kidney may a f f e c t s a l t and water e x c r e t i o n ; f o r example, increased blood flow to the medulla can lower the t o n i c i t y o f the medullary i n t e r s t i t i u m and, thereby, diminish the c a p a c i t y to concentrate u r i n e , r e s u l t i n g i n increased water e x c r e t i o n Changes i n prostaglandin synthesis a s p i r i n - l i k e drugs, o r (5_), i n f u s i o n o f a r a c h i d o n i c a c i d (8), or a d m i n i s t r a t i o n o f a loop d i u r e t i c (2), a r e l i k e l y t o be r e f l e c t e d p r i m a r i l y by a l t e r a t i o n s o f that p o r t i o n o f r e n a l blood flow which s u p p l i e s the medulla and w i l l be r e f l e c t e d by decreased o r increased blood flow t o the i n n e r and mid cortex, as measured by the d i s t r i b u t i o n of r a d i o a c t i v e microspheres w i t h i n the cortex. T h i s e f f e c t o f a l t e r e d p r o s t a g l a n d i n s y n t h e s i s on zonal d i s t r i b u t i o n o f r e n a l blood flow a r i s e s from two f a c t o r s . F i r s t , s t r a t i f i c a t i o n o f prostaglandin synthetase i n t r a r e n a l l y i s opposite t o that o f r e n i n ; the g r e a t e s t p r o s t a g l a n d i n s y n t h e t i c c a p a c i t y i s i n the p a p i l l a and medulla, the l e a s t i n the r e n a l cortex (£). I t should be noted that the apparent d i f f e r e n c e i n p r o s t a g l a n d i n b i o s y n t h e t i c c a p a c i t y between the r e n a l cortex and medulla may be r e l a t e d , i n p a r t , to the presence w i t h i n the cortex o f an i n h i b i t o r o f cyclooxygenase (10). Second, the inner c o r t i c a l and medullary c i r c u l a t i o n s are continuous, as the a f f e r e n t a r t e r i o l e s of the inner cortex extend i n t o the medulla, g i v i n g r i s e t o the vasa r e c t a (11). Therefore, changes i n p r o s t a g l a n d i n s y n t h e s i s i n the inner medulla w i l l have secondary e f f e c t s on blood flow to the outer medulla and inner and mid cortex because o f the morphol o g i c a l u n i t y o f these v a s c u l a r s t r u c t u r e s . A p o s s i b l e c l i n i c a l c o r r e l a t i o n o f these f i n d i n g s i s the nephropathy o f a n a l g e s i c abuse. Nanra e t a l have proposed that "analgesic-nephropathy" i s due to medullary ischemia secondary to reduced s y n t h e s i s o f one or more v a s o d i l a t o r p r o s t a g l a n d i n ( s ) (12), such as PGE or PGI F u r t h e r , elevated t i s s u e l e v e l s o f PGE , the presumed agent o f enhanced renomedullary blood flow, should r e s u l t from i n h i b i t i o n of PGE-9-ketoreductase. Furosemide and e t h a c r y n i c a c i d have been shown t o i n h i b i t t h i s enzyme and should thereby promote increased r e n a l blood flow to the medulla (13). The evidence f o r a p r o s t a g l a n d i n mechanism p a r t i c i p a t i n g i n the r e g u l a t i o n o f the i n t r a r e n a l d i s t r i b u t i o n o f blood flow was f i r s t obtained i n the i s o l a t e d blood-perfused kidney o f the dog 2

2

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M C G i F F AND WONG

Prostaglandins and

Renal Function

3

(14) - and l a t e r i n the conscious r a b b i t (15). One or more r e n a l p r o s t a g l a n d i n ( s ) , p r i m a r i l y PGE , i s r e s p o n s i b l e f o r mediating i n c r e a s e s i n blood flow to the r e n a l medulla i n response to s t i m u l i as d i v e r s e as s u r g i c a l trauma (5), hemorrhagic hypot e n s i o n (4), s a l t l o a d i n g (16), and loop d i u r e t i c agents (17). Those i n t e r v e n t i o n s which i n c r e a s e p r o s t a g l a n d i n production, even though they may reduce t o t a l r e n a l blood flow, can increase blood flow to the r e n a l medulla. A balanced mechanism seems to regul a t e the d i s t r i b u t i o n of r e n a l blood flow: a p r o s t a g l a n d i n mechanism i n c r e a s e s blood flow to the inner cortex and medulla, and one of the components of the r e n i n - a n g i o t e n s i n system, a n g i o t e n s i n I , probably has a major i n t r a r e n a l r o l e e f f e c t i n g decreases i n blood flow to the medulla (18). T h i s a c t i o n on the i n t r a r e n a l d i s t r i b u t i o n of blood flow may be unique f o r angiot e n s i n I , as a n g i o t e n s i n I I u s u a l l y r e s u l t s i n r e d u c t i o n i n r e n a l blood flow to a l l zone of a n g i o t e n s i n I I , whic may cause an i n c r e a s e i n medullary blood flow despite a d e c l i n e i n t o t a l r e n a l blood flow. As d i u r e t i c agents have the c a p a c i t y to a c t i v a t e the r e n i n - a n g i o t e n s i n system consequent to r e d u c t i o n of e x t r a c e l l u l a r f l u i d volume, some of the e f f e c t s on r e n a l hemodynamics may operate through t h i s mechanism. In c o n t r a s t to i t s e f f e c t on the s u r g i c a l l y - s t r e s s e d anest h e t i z e d dog, indomethacin d i d not a f f e c t r e n a l blood flow i n the conscious r e s t i n g dog, even i n doses having major t o x i c e f f e c t s ( 5 ) . T h i s f i n d i n g supports the proposal t h a t , under physiol o g i c a l c o n d i t i o n s , those mechanisms i n v o l v i n g r e n a l p r o s t a glandins are quiescent, r e q u i r i n g a noxious stimulus to be a c t i v a t e d . T h i s proposal a l s o i s i n agreement with the general c o n c l u s i o n that prostaglandins subserve a defensive f u n c t i o n , and that t h e i r r e l e a s e from an organ represents s y n t h e s i s on demand, as prostaglandins are not s t o r e d (1). Although t h i s c o n c l u s i o n appears v a l i d f o r many t i s s u e s , i t f a i l s to e x p l a i n the b a s a l e f f l u x , a l b e i t low, of prostaglandins from kidneys of the cons c i o u s r e s t i n g dog, which i s unaffected by high doses of indomethacin C5). Further, i n the conscious r a b b i t (15) and perhaps i n r e s t i n g man (19), i n h i b i t i o n of p r o s t a g l a n d i n s y n t h e s i s has been shown to r e s u l t i n increased v a s c u l a r r e s i s t a n c e . In the conscious r a b b i t , indomethacin increased r e n a l v a s c u l a r r e s i s tance two-fold, a s s o c i a t e d with a s h i f t of r e n a l blood flow to the outer cortex (15). Thus, the a c t i v i t y of i n t r a r e n a l p r o s t a g l a n d i n mechanisms i n the conscious animal under p h y s i o l o g i c a l c o n d i t i o n s may vary with the s p e c i e s . N a s j l e t t i et a l (20) have obtained evidence that the r e l e a s e of prostaglandins from the kidney under r e s t i n g c o n d i t i o n s i s determined, i n l a r g e p a r t , by the a c t i v i t y of the r e n a l k a l l i k r e i n - k i n i n system. 2

PROSTAGLANDIN BIOSYNTHETIC CAPACITY OF RENAL TISSUES An a l t e r n a t i v e explanation f o r the f a i l u r e of indomethacin

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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DIURETIC AGENTS

to a f f e c t r e n a l blood flow i n the conscious r e s t i n g dog d e r i v e s from p o s s i b l e d i f f e r e n c e s i n a c c e s s i b i l i t y of a s p i r i n - l i k e compounds to p r o s t a g l a n d i n synthetase, perhaps r e f l e c t i n g v a r i a ­ t i o n i n metabolism or d i s t r i b u t i o n of the i n h i b i t o r . Another explanation i s that the cyclooxygenase v a r i e s i n i t s suscep­ t i b i l i t y to a s p i r i n - l i k e drugs depending on the t i s s u e and species; t h i s seems l e s s l i k e l y (21). Thus, the question of access of indomethacin to i t s s i t e of a c t i o n , as w e l l as s p e c i e s and t i s s u e d i f f e r e n c e s i n the e f f e c t s of indomethacin on the p r o s t a g l a n d i n s y n t h e s i z i n g machinery, must be kept i n mind. T h i s c o n s i d e r a t i o n leads to an important observation; v i z . , the c a p a c i t y to s y n t h e s i z e p r o s t a g l a n d i n s i s d i s t r i b u t e d widely among the c e l l u l a r elements of the kidney. Cyclooxygenase i s present i n at l e a s t three d i f f e r e n t t i s s u e s i n the kidney. The i n t e r ­ s t i t i a l c e l l s of the r e n a l medulla were the f i r s t to be shown to have the c a p a c i t y to synthesiz cyclooxygenase was show d i s t a l nephron and c o l l e c t i n g ducts (22); t h i s l o c a t i o n accords with the known i n t e r r e l a t i o n s h i p s of p r o s t a g l a n d i n s and ADH (23). (Figure 1). For example, i n c r e a s e d u r i n a r y c o n c e n t r a t i n g a b i l i t y i n response to ADH occurred a f t e r treatment with indomethacin (24). P r o s t a g l a n d i n s of the Ε s e r i e s have been shown to b l u n t the e f f e c t s of ADH (23) and favor the e x c r e t i o n of f r e e water. A p r o s t a g l a n d i n mechanism may c o n t r i b u t e to the a c t i o n of those d i u r e t i c agents which i n c r e a s e l e v e l s of PGE i n the r e n a l medulla. The l a t t e r could be e f f e c t e d by an a c t i o n of the d i u r e t i c agent e i t h e r on s y n t h e s i s of p r o s t a g l a n d i n s , or on the enzyme 15-hydroxyprostaglandin dehydrogenase which degrades PGE or by i n h i b i t i o n of PGE-9-ketoreductase which transforms PGE to PGF- . Indeed, furosemide may have e f f e c t s on each of these mechanisms; i t i n c r e a s e s p r o s t a g l a n d i n s y n t h e s i s by promoting a r a c h i d o n i c a c i d d e l i v e r y to the cyclooxygenase (25) and i n h i b i t s both the dehydrogenase and reductase (13). The NADP -dependent form of the dehydrogenase has been suggested to be i d e n t i c a l to the PGE-9-ketoreductase (26). 2

2

2

POSSIBLE CIRCULATING PROSTAGLANDINS There i s l i t t l e evidence that prostaglandins f u n c t i o n as c i r c u l a t i n g hormones. An exception to t h i s was thought to be PGA which, when i n f u s e d i n t r a v e n o u s l y , was not destroyed on passage across the pulmonary c i r c u l a t i o n (31). However, i n a l l p r o b a b i l i t y , PGA i s an a r t i f a c t r e s u l t i n g from spontaneous breakdown of PGE during e x t r a c t i o n and p u r i f i c a t i o n of t i s s u e s or plasma; recent s t u d i e s based on h i g h l y s e n s i t i v e and s p e c i f i c mass spectrometric methods d i d not detect PGA i n the blood (32). Recently, P G I has been suggested to f u n c t i o n as a c i r c u l a t i n g hormone because i t s vasodepressor a c t i v i t y i s undiminished by passage across the lung (33)• 2

2

?

2

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In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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M C G i F F AND WONG

EFFERENT ARTERIOLE

Figure 1.

Prostaglandins and Renal Function

5

AFFERENT ARTERIOLE

Prostaglandin-kinin interaction in the nephron.

The generation of kinins in the distal nephron and collecting ducts results in the release of prostaglandins which inhibit the effect of ADH and thereby participate in the excretion of solute-free water. Prostaghndin-15-hydroxydehydrogenase (PGHD), PGE-9-ketoreductase (9 KR).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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DIURETIC AGENTS

RENAL ANATOMICAL COMPARTMENTS:

PROSTAGLANDINS

AND RENAL FUNCTION

Although cyclooxygenase i s present i n many t i s s u e s w i t h i n the kidney, the major products o f a r a c h i d o n i c a c i d metabolism, be they P G I , PGE , PGD , P G F , o r TXA (Figure 2), may be t i s s u e s p e c i f i c and, consequently, t h e i r e f f e c t s may be p r i m a r i l y r e s t r i c t e d to one compartment, such as the v a s c u l a r , tubular or interstitial. Thus, p r o s t a c y c l i n , a major product of arachidonic a c i d metabolism w i t h i n the blood v e s s e l w a l l (j$4), which together with other prostaglandins may a f f e c t the a c t i v i t y o f the r e n i n a n g i o t e n s i n system, i s p o s s i b l y destroyed l o c a l l y by the abundant p r o s t a g l a n d i n dehydrogenases o f the v a s c u l a r t i s s u e s (27). The r e n i n - a n g i o t e n s i n system i s p r i m a r i l y r e s t r i c t e d to the v a s c u l a r compartment as i s p r o s t a c y c l i n . T h i s i s i n c o n t r a s t to k a l l i k r e i n - k i n i n and PGE which are mainly a s s o c i a t e d with the u r i n a r y and i n t e r s t i t i a l compartments g l a n d i n synthetase w i t h i the u r i n a r y compartment, p a r t i c u l a r l y the d i s t a l nephron and c o l l e c t i n g ducts (22), f a c i l i t a t e s the i n t e r a c t i o n o f p r o s t a glandins with k i n i n s and ADH. For example, entry o f k a l l i k r e i n i n t o the d i s t a l tubules, and subsequent formation o f k i n i n s , r e s u l t s i n r e l e a s e o f one or more prostaglandins by k i n i n s from s i t e s o f p r o s t a g l a n d i n generation along the c o l l e c t i n g ducts. I n h i b i t i o n o f the e f f e c t s o f ADH can occur, then, i n response to the kinin-mediated generation o f prostaglandins i n the d i s t a l nephron; t h i s r e s u l t s i n the e x c r e t i o n o f s o l u t e - f r e e water. A recent study by Weber et a l (J36) i n d i c a t e s that the a c t i v i t y o f a major p r o s t a g l a n d i n metabolizing enzyme (37), PGE-9-ketoreductase, which converts PGE t o PGF , i s i n f l u e n c e d by s a l t i n t a k e . Thus, reabsorption o f water i s f a c i l i t a t e d by increased a c t i v i t y of t h i s enzyme, which has the e f f e c t o f lowering l e v e l s o f PGE i n t r a r e n a l l y by f a v o r i n g formation o f P G F . As P G F , u n l i k e PGE , does not i n h i b i t ADH, increased a c t i v i t y o f PGE-9-ketoreductase w i l l f a c i l i t a t e reabsorption i n water. The "loop d i u r e t i c " agents have already been noted to be capable o f i n h i b i t i n g the a c t i v i t y o f t h i s enzyme. I t should be noted that k i n i n s , i n a d d i t i o n to promoting p r o s t a g l a n d i n s y n t h e s i s , are a l s o capable o f i n c r e a s i n g the a c t i v i t y o f PGE-9-ketoreductase (38), and that these e f f e c t s may be c r u c i a l to the a b i l i t y o f k i n i n s to a l t e r e x c r e t i o n o f s o l u t e - f r e e water as a f f e c t e d by the s t a t e o f sodium balance. As i n h i b i t i o n o f p r o s t a g l a n d i n synt h e s i s has been shown to prevent increased f r e e water generation induced by b r a d y k i n i n (39), a p r o s t a g l a n d i n mechanism appears t o be necessary f o r t h i s e f f e c t o f the k i n i n (Figure 1). 2

2

2

2a

2

2

2

2

2

2a

2a

2

PROSTAGLANDINS

AND SALT EXCRETION

The concept o f segregation o f cyclooxygenases w i t h i n s e v e r a l f u n c t i o n a l compartments o f the kidney and d i f f e r e n t p r o s t a glandins a r i s i n g from these compartments i s u s e f u l f o r

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 2.

Metabolism of arachidonic acid by the prostaglandin synthetase complex. t

t

t

Major products of vascular tissues are prostacyclin (PGI ) and PGE ; a major product of blood platelets is thromboxane A

g

(TxA )

-4

I §

1

ι

ι

ι

!

i

Î

DIURETIC AGENTS

8

i n t e r p r e t i n g the v a r i a b l e e f f e c t s of one or more p r o s t a g l a n d i n mechanisms on s a l t e x c r e t i o n . The n a t r i u r e t i c e f f e c t of e i t h e r the p r i n c i p a l r e n a l p r o s t a g l a n d i n , PGEp, or i t s precursor, a r a c h i d o n i c a c i d , cannot be d i s s o c i a t e d e a s i l y from i t s e f f e c t s on the r e n a l c i r c u l a t i o n . In the only i n vivo study which examined the e f f e c t of PGE on t u b u l a r f u n c t i o n uncomplicated by v a s c u l a r e f f e c t s , Kauker, u s i n g m i c r o - i n j e c t i o n techniques, demonstrated that i n t r a l u m i n a l i n j e c t i o n o f PGEp i n r a t s r e s u l t e d i n i n h i b i t i o n of sodium r e a b s o r p t i o n (40). F u r t h e r , Stokes and Kokko demonstrated an i n h i b i t o r y e f f e c t o f PGE on sodium t r a n s port i n i s o l a t e d perfused r e n a l c o l l e c t i n g tubules of r a b b i t s p r e t r e a t e d with m i n e r a l o c o r t i c o i d s (41). In conscious r a t s , N a s j l e t t i et a l (20) demonstrated that m i n e r a l o c o r t i c o i d t r e a t ment not only increased k a l l i k r e i n e x c r e t i o n , but a l s o enhanced e x c r e t i o n of PGEp by two- to t h r e e - f o l d Augmented e x c r e t i o n o f k a l l i k r e i n and PGE i n the s a l t and water r e t a i n i n the r a t , i n h i b i t i o n of p r o s t a g l a n d i n s y n t h e s i s a l s o r e s u l t s i n i n c r e a s e d concentration of sodium c h l o r i d e i n the r e n a l medulla (42). The l a t t e r suggests that exaggerated t u b u l a r r e a b s o r p t i o n of sodium i n the ascending limb of the loop of Henle r e s u l t s from e l i m i n a t i n g a p r o s t a g l a n d i n mechanism which promotes s a l t excret i o n . On the other hand, i n the conscious dog undergoing a water-induced d i u r e s i s , both indomethacin and meclofenamate have been reported to increase sodium e x c r e t i o n (43). A possible p r o s t a g l a n d i n mechanism which prevents demonstration of the d i r e c t n a t r i u r e t i c a c t i o n of b r a d y k i n i n was described by McGiff et a l i n the blood-perfused i s o l a t e d canine kidney (39). Thus, a n a t r i u r e t i c a c t i o n of b r a d y k i n i n was not shown u n t i l p r o s t a g l a n d i n s y n t h e s i s was i n h i b i t e d by indomethacin. These seemingly d i s c r e p a n t s t u d i e s may be r e c o n c i l e d i f i t i s recognized that the experimental c o n d i t i o n s determine not only the l e v e l of p r o s t a g l a n d i n a c t i v i t y , but a l s o the major s p e c i e s of p r o s t a g l a n d i n s produced w i t h i n the u r i n a r y compartment. As these vary, the e f f e c t s o f indomethacin, which a l s o a l t e r s p r o s t a g l a n d i n metabol i z i n g enzymes (13), w i l l depend on the l e v e l and p r o f i l e of prostaglandins produced under a given set of c o n d i t i o n s . T h i s , i n t u r n , i s r e l a t e d to the s t a t e of s a l t and water balance, the degree of s t r e s s occasioned by a n e s t h e s i a and surgery, the a c t i v i t y of other hormonal systems, the " i n t r i n s i c " a c t i v i t y o f the cyclooxygenase as determined by n a t u r a l i n h i b i t o r s and a c t i v a t o r s , and, f i n a l l y , the s p e c i e s being s t u d i e d . These general c o n s i d e r a t i o n s f o r c e the c o n c l u s i o n that the products of cyclooxygenases i n the v a r i o u s compartments w i t h i n the kidney may vary with experimental c o n d i t i o n s , as w e l l as i n h e a l t h and i n d i s e a s e . For example, thromboxane, a powerful vasoconstrictor; i s not normally synthesized by the kidney. However, when r e n a l f u n c t i o n i s d i s t u r b e d , as by acute u r e t e r a l l i g a t i o n , thromboxane s y n t h e s i s may occur (44). I t s production may c o n t r i b u t e to the l a t e i n c r e a s e i n r e n a l v a s c u l a r r e s i s t a n c e i n response to 2

2

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1. MC GIFF

AND

WONG

Prostaglandins and Renal Function

9

ureteral obstruction (45 ). Changes in extracellular potassium concentration can also affect renal prostaglandin synthesis (46 ). As urinary k a l l i k r e i n concentrations have been positively correlated with excretion of potassium, but not sodium (47), the possibility of a potassiumdependent interaction of prostaglandins with kallikrein-kinins should be considered. Thus, induction of potassium deficiency has been shown to result i n enhanced renal prostaglandin synthesis (48). Hyposthenuria associated with potassium deficiency, then, may be related to inhibition of the effects of ADH (23) consequent to increased production of PGE^ or a related prostaglandin. SUMMARY Those diuretic agent primary sites of actio g p and the cortical collecting ducts, where they have a primary effect on chloride transport (j\9), can be shown to have major effects not only on the renin-angiotensin system (6), but also on the k a l l i k r e i n - k i n i n (50) and prostaglandin systems (13). There i s evidence suggesting that their diuretic action may be related partially to an effect on the vasodilator-diuretic system of the kidney, the kallikrein-kinin-prostaglandin system. Thus, aspirin-like compounds have been shown to blunt the diuretic action of furosemide (j51), although this effect of antiinflammatory acids is complicated by their inhibition of the organic acid secretory system. Integrity of the latter may be required for access of these diuretic agents to their active sites. Further, furosemide and ethacrynic acid have been shown to inhibit two of the major prostaglandin catabolizing enzymes, prostaglandin-15-hydroxydehydrogenase and PGE-9-ketoreductase (13). Their effects on these enzymes may result in increased levels of PGE and PGI which may then contribute to vasodilator-diuretic mechanisms. The design of agents which have major effects on prostaglandin metabolism is well underway and has already resulted in novel diuretic agents (52). Acknowledgments - We thank Mrs. Cathy Reynolds and Mrs. Sue Hatton for assistance in typing the manuscript. This study was supported by USPHS Grants HL-18845 and HL-22075 and American Heart Association Grant 77-987. Literature Cited 1. Änggard, Ε . , Bohman, S. O., G r i f f i n , J . Ε., III, Larsson, C., and Maunsbach, A. B . , Acta Physiol. Scand. (1972), 84, 231-246. 2. Olsen, U. B . , Acta Pharmacol. Toxicol. (1977), 41, 1-31. 3. McGiff, J . C., Crowshaw, Κ., Terragno, Ν. Α . , and Lonigro, Α . , Nature (1970), 227, 1255-1257. 4. Vatner, S. F., J . C l i n . Invest. (1974), 54, 225-235. 5. Terragno, Ν. Α . , Terragno, D. A. and McGiff, J . C. Circ. Res. (1977), 40, 590-595. 2

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

10

DIURETIC

AGENTS

6. B a i l i e , M.D., Davis, L . E . and Loutzenhiser, R . , Am. J . Physiol (1973), 224, 425-430. 7. Itskovitz, H. D . , Terragno, N. A. and McGiff, J. C . , Circ. Res. (1974), 34, 770-776. 8. Chang, L . C. T., Splawinski, J . Α . , Oates, J. A. and Nies, A. S., C i r c . Res. (1975), 36, 204-207. 9. Larsson, C. and Änggard, E., Eur. J. Pharmacol. (1974), 21, 30-36. 10. Terragno, Ν. Α . , McGiff, J. C. and Terragno, Α . , C l i n . Res. (1978), 26, 545A (abstract). 11. Fourman, J. and Moffat, D. B . , "Blood Vessels of the Kidney", p. 58, Oxford, Blackwell Scientific Publications, Oxford, England, 1971. 12. Nanra, R. S., Chirawong, P. and Kincaid-Smith, P . , Aust. N. Z. Med. (1973), 3, 580-586 13. Stone, K. J. and Hart 197-207. 14. Itskovitz, H. D . , Stemper, J., Pacholczyk, D. and McGiff, J. C., C l i n . S c i . (1973), 45, 321s-324s. 15. B e i l i n , L . J. and Bhattacharya, J., J. Physiol. (1977), 269, 395-405. 16. Papanicolaou, N . , Safar, Μ., Hornych, Α . , Fontaliran, F., Weiss, Υ . , Bariety, J. and M i l l i e z , P . , C l i n . S c i . Molec. Med. (1975), 49, 459-463. 17. Olsen, U. B. and Ahnfelt-Ronne, I . Acta Physiol. Scand. (1976), 97, 251-257. 18. Itskovitz, H. and McGiff, J. C., Circ. Res. (1974), 34-35, (Suppl I ) , 65-73. 19. Wennmalm, Α . , IRCS (1974), 2, 1099. 20. Nasjletti, Α . , McGiff, J . C. and Colina-Chourio, J., Circ. Res., i n press. 21. Pong, S. S. and Levine, L., J. Pharmacol. Exp. Ther. (1976), 196-197, 226-230. 22. Smith, W. L . and Wilkin, G. P . , Prostaglandins (1977), 13, 873-892. 23. Grantham, J. J. and Orloff, J., J. C l i n . Invest. (1968), 47, 1154-1161. 24. Berl, P . , Raz, Α . , Wald, H . , Horowitz, J. and Czaczkes, W., Am. J. Physiol. (1977), 232, F529-F537. 25. Weber, P. C., Scherer, B. and Larsson, C., Eur. J . Pharmacol. (1977), 41, 329-332. 26. Hassid, A. and Levine, L., Prostaglandins (1977), 13, 503-516. 27. Wong, P. Y - K . , Sun, F . F . and McGiff, J . C., J. B i o l . Chem. (1978), i n press. 28. Larsson, C., Weber, P. and Änggard, E., Eur. J. Pharmacol. (1974), 28, 391-394. 29. Gerber, J. G . , Branch, R. Α . , Nies, A. S., Gerkens, J. F., Shand, D. G . , H o l l i f i e l d , J. and Oates, J. Α . , Prostaglandins (1978), 15, 81-88.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1.

MCGIFF

AND WONG

Prostaglandins and Renal Function

11

30. Terragno, D. Α . , Crowshaw, K., Terragno, N. A. and McGiff, J. C . , C i r c . Res. (1975), 36-37, (Suppl I ) , 76-80. 31. McGiff, J. C., Terragno, Ν. Α . , Strand, J. C., Lee, J. B . , Lonigro, A. J. and Ng, Κ. K. F., Nature (1969), 223, 742-745. 32. F r o l i c h , J. C., Sweetman, B. J., Carr, Κ., H o l l i f i e l d , J. W. and Oates, J. Α . , Prostaglandins (1975), 10, 185-195. 33. Armstrong, J. Μ., Lattimer, N . , Moncada, S. and Vane, J. R . , Br. J. Pharmac. (1978), 62, 125-130. 34. Moncada, S., Gryglewski, R. J., Bunting, S. and Vane, J. R., Prostaglandins (1976), 12, 715-737. 35. Carretero, O. A. and Scicli, A. G . , Fed. Proc. (1976), 35, 194-198. 36. Weber, P. C., Larsson, C. and Scherer, B . , Nature (1977), 266, 65-66. 37. Leslie, C. A. and Levine L., Biochem Biophys Res Commun (1973), 52, 717-724. 38. Wong, P. Y-K, Terragno, , Terragno, , J. C., Prostaglandins (1977), 13, 1113-1125. 39. McGiff, J. C., Itskovitz, H. D. and Terragno, Ν. Α . , C l i n . S c i . Molec. Med. (1975), 49, 125-131. 40. Kauker, M. L., Proc. Soc. Exp. B i o l . Med. (1977), 154, 272-277. 41. Stokes, J. B. and Kokko, J. P . , J. C l i n . Invest. (1977), 59, 1099-1104. 42. Ganguli, M . , Tobian, L., Azar, S. and O'Donnell, Μ., Circ. Res. (1977), 40, (Suppl I ) , 135-139. 43. Kirschenbaum, M. A. and Stein, J. Η . , J. C l i n . Invest. (1976), 57, 517-521. 44. Morrison, Α . , Nishikawa, K. and Needleman, P . , Nature (1977), 267, 259-260. 45. Yarger, W. E . and G r i f f i t h , L . D . , Am. J. Physiol. (1974), 227, 816-826. 46. Zusman, R. M. and Reiser, H. R., J. C l i n . Invest. (1977), 60, 215-223. 47. Zinner, S. H., Margolius, H. S., Rosner, B . , Reiser, H. R. and Kass, Ε. H., Am. J. Epidem. (1976), 104, 124-132. 48. Galvez, O. G . , Bay, W. H., Roberts, B. W. and F e r r i s , R. F., Circ. Res. (1977), 40, (Suppl 9), 11-16. 49. Rocha, A. S. and Rokko, J. P . , J. C l i n . Invest. (1973), 52, 612-623. 50. Croxatto, H. R., Roblero, J. S., Garcia, R. L., Corthorn, J. H. and San Martin, M . , Acta Physiol. Latino Am. (1973), 22-23, 556-558. 51. Lee, J. B . , Proc. 6th Int. Congr. Nephrol. (1975), 1, 348-354. 52. Cragoe, E . J., Jr., Schultz, Ε. Μ., Schneeberg, J. D . , Stokker, G. E., Woltersdorf, O. W., Jr., F a n e l l i , G. Μ., Jr., and Watson, L . S., J. Med. Chem. (1975), 18, 225-228. RECEIVED

August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2 Structure-Activity Relationships of Aminobenzoic Acid Diuretics and Related Compounds (1) O. B. TVAERMOSE NIELSEN and P. W. FEIT Leo Pharmaceutical Products, 2750 Ballerup, Denmark

In a series of paper presented some of our results based on a more or less systematic structural alteration of the sulfamoylbenzoic acid diuretics in order to elucidate the structural requirements for high-ceiling diuretic a c t i v i t y . When we started this work, for reasons previously discussed (3), the only existing high-ceiling sulfonamide diuretics were the N-substituted 4-chloro-5-sulfamoylanthranilic acids represented by furosemide (Formula 1). Although the diuretic characteristics of furosemide were quite different from those of the thiazides or the thiazide-type diuretics, thereby reflecting a different site of action in the nephron, i t still shared structural features with the latter compounds. Thus, furosemide f i t the structural requirements for diuretic activity of the benzenesulfonamides and related bicyclic compounds originally proposed by Sprague (11). He predicted that an unsubstituted sulfamoyl group, an activating group represented mainly by chloro and trifluoromethyl and an electronegative substituent (which might be part of a condensed ring system) should be present in the molecule and have the mutual positions reflected in furosemide.

Our f i r s t approach was to move the substituted amino function (which is not part of Sprague's predictions) to the 3-position, with almost complete retention of diuretic activity (2). More surprisingly, replacement of the chlorine atom by various other groups in both the 2-substituted (4, 5) and the 3-substituted 0-8412-0464-0/78/47-083-012$05.00/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

NIELSEN

AND

13

Aminobenzoic Acid Diuretics

FEIT

(3, 6, 7, 8) sulfamoylbenzoic a c i d d i u r e t i c s , was found to enhance the d i u r e t i c potency. The most potent compounds (Formula 2) were those i n which an unsubstituted or s u b s t i t u t e d phenyl group was l i n k e d to the 4 - p o s i t i o n by a group X, which represents NH, 0, S, SO, S0 , CH or CO. These 4-substituents are not merely new a c t i v a t i n g groups" i n the sense t h a t Sprague described, s i n c e replacement of chloro by phenoxy or phenylthio i n some s e l e c t e d t h i a z i d e s and t h i a z i d e - t y p e d i u r e t i c s l e d to compounds which were not d i u r e t i c (12). The p r e d i c t i o n s of Sprague, although s t i l l of value as f a r as the t h i a z i d e - t y p e d i u r e t i c s were concerned, were found not to account f u l l y f o r the s t r u c t u r a l requirements of the high-ceiling diuretics. 2

2

11

X = NH,

0, S, SO,

S0 ,

η-butyl, benzyl, methyl or

2

CH , 2

CO

furyl-

thienylmethyl

When placed i n the 3-position, many v a r i a t i o n s of the sub­ s t i t u e n t R i n formula 2 c o n t r i b u t e to high potency. However, when l o c a t e d i n the 2 - p o s i t i o n , the only a c t i v e compounds are those where R i s r e s t r i c t e d to a s u b s t i t u t e d amino f u n c t i o n , p r e f e r a b l y the 2-furylmethylamino group. Bumetanide (Formula 3) was s e l e c t e d f o r f u r t h e r i n v e s t i g a t i o n and shown (13) to be 40 to 60 times more potent than furosemide; t h i s was a l e v e l of potency e x h i b i t e d i n the dog assay by many compounds of formula 2. I t s u t i l i t y i n c l i n i c a l p r a c t i c e has since been e s t a b l i s h e d (14). NHCH CH CH CH 2

H N0 S ' 2

2

2

3

COOH

2

3 The compounds of formula 2, i n which X represents carbonyl and R a s u b s t i t u t e d amino f u n c t i o n , are not i s o l a b l e due to a pHdependent e q u i l i b r i u m i n aqueous s o l u t i o n . At p h y s i o l o g i c a l pH, the e q u i l i b r i u m favors the cyclodehydration products, i . e . , the b e n z i s o t h i a z o l e dioxides of formula 4. I t has been suggested (5>, 6), however, that the potent d i u r e t i c a c t i v i t y (one f o u r t h to one tenth of that of bumetanide) observed a f t e r a d m i n i s t r a t i o n of these b e n z i s o t h i a z o l e s to dogs i n v o l v e s the i n t e r a c t i o n of the corresponding 4-benzoylsulfamoylbenzoic a c i d s (Formula 5) with

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

14

DIURETIC

AGENTS

the r e c e p t o r which i s p o s s i b l e because of the dynamic e q u i l i b r a t i o n i n plasma. Thus, the potency of the b e n z i s o t h i a z o l e dioxides (Formula H) appear e r t y . For the correspondin 5-sulfamoylbenzoic a c i d s , the e q u i l i b r i u m i s t o t a l l y to the s i d e of the benzoyl compound (8); t h i s might e x p l a i n why compound 6 (Table I) i s one of the most potent, h i g h - c e i l i n g d i u r e t i c s that we synthesized. Since t h i s compound shows s i g n i f i c a n t d i u r e t i c a c t i v i t y i n the dog assay a f t e r an o r a l dose of only 1 yg/kg, i t i s 5 to 10 times more potent than bumetanide ( 8_).

TABLE I u r i n a r y E x c r e t i o n i n a 3 hr. P e r i o d f o l l o w i n g I.V. A d m i n i s t r a t i o n of Compound 6 to Dogs, Expressed f o r V o l . i n ml/kg and f o r E l e c t r o l y t e s i n mEq/kg.

Treatment mg/kg

control

0.001 0.01 0.1

Vol.

1 k

25 38

Na

+

0.10 0.7 2.5 h.k

K

+

o.i6 0.28 0.53 0.97

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Cl"

o.o8 0.7 3-5 *.9

2.

NIELSEN

15

Aminobenzoic Acid Diuretics

A N D FETT

f^*yS>£

X = NH, 0, S, CO Il

1

1

R = NHR , OR , SR

1

i n which R

H a C O s S ^ ^ ^ COOH

preferably i s n-butyl,

Y

benzyl or furylmethyl

1

Another d i s c o v e r y was that a c t i v e d i u r e t i c s of the type represented by formula 7 could be obtained when the sulfamoyl group was replaced by the s t e r i c a l l y s i m i l a r mesyl group (°J · Various 3-substituents c o n t r i b u t e to the comparatively high potency o f these compounds s t i t u e n t allowed i s th t i o n s encouraged the i n v e s t i g a t i o n o f the i n f l u e n c e o f other s u b s t i t u e n t s i n the 5 - p o s i t i o n . A s u i t a b l y s u b s t i t u t e d amino f u n c t i o n , p r e f e r a b l y a formamido group i n place o f the sulfamoyl group (Formula 8), l e d to a c t i v e d i u r e t i c s (10). 0, S, CH , CO 2

1

1

1

1

NHR , OR , SR , CHsR i n which R p r e f e r a b l y i s η-butyl, b e n z y l , f u r y l ­ methyl o r t h i e n y l m e t h y l . 1

H, lower a l k y l , NEfe, NH-lower a l k y l ,

lower

alkoxy

Table I I shows the data obtained with one o f the most potent compounds with t h i s p a r t i c u l a r s t r u c t u r a l a l t e r a t i o n . Compound 9 was found to be approximately one tenth as potent as bumetanide. D i s a p p o i n t i n g l y , the e l e c t r o l y t e e x c r e t i o n p a t t e r n was s t i l l s i m i l a r to that o f bumetanide. The corresponding 2,4disubstituted-5-acylaminobenzoic a c i d s were, however, i n a c t i v e . X = NH, 0, S, CO 1

1

R = NHR , OR , SR

1

i n which

p r e f e r a b l y i s η-butyl o r benzyl R2 = OH, NHg, NH-lower a l k y l

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

R

1

DIURETIC

16

AGENTS

TABLE I I U r i n a r y E x c r e t i o n (Average o f 3 Values) i n a 3 n r . Period f o l l o w i n g I.V. A d m i n i s t r a t i o n o f Compound 9 to Dogs, Expressed f o r V o l . i n ml/kg and f o r E l e c t r o l y t e s i n mEq/kg.

HJjHN^^/^COOH 0

9 Treatment mg/kg

control

0.1 0.25 1.0 5.0

Vol.

1 9 18 22 32

Na

Cl"

+

0.10 1.0 2.1 2.5 3.7

0.16 0.2k

οΛι 0.49 0.83

0.08 1-3 2.6 3.1

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

NIELSEN AND

FEiT

Aminobenzoic Acid Diuretics

17

In a d d i t i o n , i s o p h t h a l i c a c i d d e r i v a t i v e s o f formula 10 were prepared (15). Some o f them exerted moderate d i u r e t i c a c t i v i t y ; however, again, the c h a r a c t e r i s t i c e l e c t r o l y t e e x c r e t i o n p r o f i l e of bumetanide was observed. The above b r i e f survey o f p r e v i o u s l y reported r e s u l t s o f s t r u c t u r a l m o d i f i c a t i o n s i n benzoic a c i d d i u r e t i c s r e v e a l s that our e f f o r t s had been d i r e c t e d toward compounds i n which the c a r b o x y l i c a c i d f u n c t i o n remained i n t a c t . I f such a f u n c t i o n i s present i n b i o l o g i c a l l y a c t i v e molecules, i t s a c i d i c character u s u a l l y has, f o r known reasons, an e f f e c t on a c t i v i t y and/or potency. Therefore, i t was considered to be o f i n t e r e s t to i n v e s t i g a t e whether a carboxyl f u n c t i o n i s a p r e r e q u i s i t e f o r high-ceiling diuretic activity. Consequently, d e r i v a t i v e s i n which t h i s group was replaced by other f u n c t i o n s were synthesized. 3,4-Disubstituted-5-sulfamoylbenzenesulfini acid d sul f o n i c a c i d s o f formula corresponding c a r b o x y l i y means o f a C u r t i u s degradation f u r n i s h e d the corresponding a n i ­ lines. The l a t t e r d e r i v a t i v e s were converted to the s u l f o n y l c h l o r i d e s v i a d i a z o t i z a t i o n and the Meerwein-reaction. These were subsequently hydrolyzed to s u l f o n i c a c i d s or reduced to s u l f i n i c a c i d s . I t can be seen from Table I I I that these rather strong acids possess potent d i u r e t i c a c t i v i t y . Their d i u r e t i c p r o f i l e i s q u i t e s i m i l a r to that o f bumetanide; however, they have a s h o r t e r duration o f a c t i o n . The corresponding" 2 , 4 , 5 - t r i s u b s t i t u t e d a c i d s were not i n v e s t i g a t e d s i n c e , 15 years p r e v i o u s l y , the s u l f i n i c a c i d analogue o f furosemide had been found by us to be devoid o f a c t i v i t y a t a dose o f 10 mg/kg i n the dog assay. In c o n t r a s t , 3-butylamino-4-chloro-5-sulfamoylbenzenesulfinic a c i d was found to be d i u r e t i c a t a dose o f 1 mg/kg. A s e r i e s o f 3,4-disubstituted-5-sulfamoylbenzylamines was synthesized (16). In the dog assay, many of these compounds e x h i b i t e d h i g h - c e i l i n g d i u r e t i c a c t i v i t y i n the range o f potency from that o f furosemide to that o f bumetanide. Therefore, an extended s e r i e s o f benzylamines o f formula 12 (see Table IV) having the 3-, 4-»·, and 5-substituents o f bumetanide was preparecj i n order to e l u c i d a t e the i n f l u e n c e o f the N-substituent(s) i n the benzylamine moiety on d i u r e t i c a c t i v i t y . S e l e c t e d members, o f t h i s s e r i e s are tabulated i n Table IV. I t appears that many R and R s u b s t i t u e n t s , such as hydrogen, lower s t r a i g h t chain a l k y l , a l l y l , benzyl, f u r y l m e t h y l , pyridylmethyl and unsubstituted as w e l l as s u i t a b l y s u b s t i t u t e d phenyl, c o n t r i b u t e to high potency. Disubs t i t u t i o n , as exemplified by N,N-dimethyl, Ν,Ν-diethyl and some N - a l k y l , N-hydroxyethyl d e r i v a t i v e s , a l s o i s an allowed s t r u c t u r a l m o d i f i c a t i o n . I n c o n t r a s t , i n c o r p o r a t i o n o f a n i t r o g e n atom i n a r i n g system to provide d e r i v a t i v e s o f p i p e r i d i n e , morpholine and Ν'-substituted piperazine r e s u l t s i n compounds which are i n a c t i v e at the doses t e s t e d . Likewise, s u b s t i t u t i o n with branched a l k y l , a c y l , carbamoyl or N-substituted carbamoyl groups g e n e r a l l y decreases or abolishes d i u r e t i c a c t i v i t y .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

18

TABLE I I I U r i n a r y E x c r e t i o n i n a 6 n r . Period f o l l o w i n g I.V. A d m i n i s t r a t i o n to Dogs o f 1 mg/kg Expressed f o r V o l . i n ml/kg and f o r E l e c t r o l y t e s i n mEq/kg.

H

11 η

Control (extreme values from 108 exp.)

Vol.

1

Cl

k.2

0.05 0Λ5

O.3O

0.06 0.28

37

4.1

1.02

5-3

4.7

1.05

4.5

0.3-

2

Na"

0.06

0

NHC Ife

0

NHCH C H5

2

0

SC H9 4

2

29

4.0

1.15

1.7

0C4lfe

2

34

3.0

1.31

5-2

0

NHC He

3

22

2.6

0.62

3.1

0

NHCH C H5

3

48

4.3

0.91

6.6

0

•a

3

35

3.3

0.91

4.1

CH

4

2

2

e

4

2

e

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

NIELSEN AND

FEiT

Aminobenzoic Acid Diuretics

19

Various 3- and 4-substituents, found i n the s e r i e s of benzoic a c i d s of formula 2 to c o n t r i b u t e to h i g h - c e i l i n g potency, were incorporated i n a s e r i e s of compounds i n which the v a r i a t i o n of the benzylamine moiety was r e s t r i c t e d to s e l e c t e d N - s u b s t i t u e n t s . Since the i n f l u e n c e of these N-substituents d i d not deviate from that observed i n the s e r i e s of bumetanide analogues, only the r e s u l t s obtained with compounds of formula 13 having the benzylamino n i t r o g e n atom s u b s t i t u t e d by phenyl are given i n Table V. As p r e v i o u s l y observed with the.benzoic a c i d d i u r e t i c s of formula 2, groups such as R 0, R S or R NH (where R i s η-butyl, benzyl, f u r y l m e t h y l or thienylmethyl) placed i n the 3-position enhanced the d i u r e t i c a c t i v i t y . Likewise, phenyl attached to the 4p o s i t i o n through the well-known l i n k s , NH, 0, S or CHp, a l s o was found to c o n t r i b u t e to the potent h i g h - c e i l i n g d i u r e t i c a c t i v i t y of the benzylamine s e r i e s A number of 2,4-disubstituted-5-sulfamoylbenzylamine as the 2-substituent th furosemide and r e l a t e d a n t h r a n i l i c a c i d s were found to be i n a c ­ t i v e . T h i s l a c k of a c t i v i t y might w e l l be due to an i n a d m i s s i b l e s t r u c t u r a l m o d i f i c a t i o n . However, a f t e r a d m i n i s t r a t i o n of se­ l e c t e d 3-substituted benzylamines to dogs, the presence of the corresponding benzoic a c i d s i n the urine has been demonstrated qualitatively. Consequently, the question a r i s e s as to whether the d i u r e t i c response f o l l o w i n g treatment with 3-substituted benzylamines should be explained s o l e l y on the b a s i s of metabolic transformation to the corresponding benzoic a c i d d i u r e t i c s of formula 2. The marked d i f f e r e n c e i n d i u r e t i c a c t i v i t y between the 2- s u b s t i t u t e d and the 3-substituted benzylamines could be due only to the repressed metabolism of the 2 - d e r i v a t i v e s due to the s t e r i c e f f e c t of the o r t h o - s u b s t i t u e n t . On the other hand, the above mentioned e f f e c t s on d i u r e t i c a c t i v i t y may be accounted f o r by the s i m i l a r d i f f e r e n c e s between the 2- and the 3-substituted d e r i v ­ a t i v e s i n the 5-acylaminobenzoic s e r i e s and between the 2- and 3- substituted-4-chloro-5-sulfamoylbenzenesulfinic a c i d s e r i e s . Furthermore, the i n c o n s i s t e n c y between the observed d i u r e t i c a c t i v i t i e s and the r a t e at which the subject benzylamines (Table IV) would be expected to be metabolized, might support the view that the 3-substituted benzylamines possess i n t r i n s i c d i u r e t i c activity. The only conclusions we dare to draw from the r e s u l t s of our research during more than a decade i n the e x c i t i n g f i e l d of d i u r e t i c s are that numerous s t r u c t u r a l a l t e r a t i o n s of d i s u b s t i tuted sulfamoylbenzoic a c i d d i u r e t i c s l e d to compounds with h i g h l y potent h i g h - c e i l i n g d i u r e t i c a c t i v i t y , and that the 3>4-disubstituted members of t h i s s e r i e s are l e s s s e n s i t i v e to such a l t e r a ­ t i o n s than are the 2,4-disubstituted members. F u r t h e r e x p l o r a t i o n i n t h i s area might s t i l l , i n our o p i n i o n , uncover benzene r i n g s u b s t i t u e n t s or s u b s t i t u t i o n patterns l e a d i n g to compounds with high-ceiling diuretic activity. Acknowledgment - The authors are g r e a t l y indebted to the

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

20

DIURETIC

AGENTS

TABLE IV Urinary E x c r e t i o n (Mean o f 2 Values) i n a 6 n r . Period f o l l o w i n g I.V. (when marked with an a s t e r i s k p.o.) A d m i n i s t r a t i o n to Dogs of 1 mg/kg, Expressed f o r V o l . i n ml/kg and f o r E l e c t r o l y t e s i n mEq/kg.

R

R

2

3

Control (extreme values from 108 exp.) H methyl ethyl n-propyl isopropyl allyl CH C(CH )=CH n- b u t y l isobutyl sec-butyl tert-butyl isopentyl n-hexyl CH CH 0H CH CH0HCH CH CH NEt methyl ethyl n-propyl methyl ethyl isopropyl CH CQH5 CH C H -4-Me CH C H -3-Cl 2

3

2

2

2

3

2

2

2

2

2

e

4

2

e

4

2

H H H H H H H H H H H H H H H H methyl ethyl η-propyl CHgCI^OH CH CEfeOH CH CH 0H H H H 2

2

2

K+

Cl"

0.05 0.45

0.06 0.30

0.06 0.28

t.5

0.81 0.35

2.6 2.4 2.4 2.6 0.7 3.7 2.1 1.6 0.2 0.4 1.2 0.2 0.6 0.3 1.1 0.1 2.6 1.2 0.6 0.4 1.9 2.0

Vol.

Na

0.3 4.2 23 19 17 *17 12 29 20 8 3 5 *7 3 9 3 9 1 21 10 *4 3 13 15 33 3 *11

+

1.5 2.0 1.9 0.8 3-5 1.4 1.4

O.k o.k

1.1 0.3 1.1 0.3 1.0 0.1 2.1 1.0 0.5 0.5 1.3 1.5 3-1 0.4 1.1

0.48

0.67 0.32 1.3 0.35 0.42 0.30 0.09 0.27 0.13 0.75 0.18 0.39 0.05 0.48

0.37 0.21 0.18 0.32 0Λ7 1.02 0.16 0.24

k.5

0.5 1.4

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

NIELSEN

Table IV

AND

(Continued)

CH c H -3-0H (H C H -4-0Me CH CgH5 2

4

e

2

4

e

21

Aminobenzoic Acid Diuretics

FEiT

2

Vol.

Na

Η Η methyl

7

6

0.8 0.8 0.8

0.22

11

0.4-0

0.6 1.3 1.0

Η

19

2.8

0.86

1.9

Η

16

1.0

0.31

1.1

Η

25

2.6

0.67

2.9

Η

2

0.3

0.11

0.2

methyl

7

0.6

0.38

0.7

methyl

7

0.9

0.26

0.9

*.5

1.15 0.45 0.23 0.09 0.15

+

Κ

Cl"

R3

+

0.25

Η

CoH -2-Me C H -4-Me CQH -2-CF C H -3-F C H -3-Cl C H -4-Br C H -2-0H CeH -4-0H C H -2-0CH C H -3-0C H 4

e

4

4

e

3

4

e

4

e

4

e

4

e

4

e

4

4

3

2

5

33

Η Η Η Η Η Η Η Η Η Η Η

21

9 5 2

Ο" COCH CONH CONHCH CONHC H 3

2

3

3

T

•CH CH OH 2

Η Η Η Η

2

1.9

0.2

0.12

0.2

5

0.7

0.48

1.1

3

0.2

ο.3ΐ

0.2

3

0.4

0.22

0.3

2 1

0.3 0.1 0.7 1.0

0.14

0.1 0.1 0.7 1.0

3

3

0.7 0.2 0.3 0.2

0.2 0.2

2

Ο

Ό Ν ­ •CH

0.11

5Λ

3.7

0.37 0.14 0.39 0.32 0.18

13 6 14

c °

2.0 0.8 0.2 0.5 0.2 1.1 0.5 1.2 0.2 0.2

8 8

0.10

0.59 0.39

0.5

1.5

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

22

DIURETIC

AGENTS

TABLE V u r i n a r y E x c r e t i o n (Mean o f 2 Values) i n a 6 h r . P e r i o d f o l l o w i n g I.V. A d m i n i s t r a t i o n to Dogs o f 1 mg/kg, Expressed f o r V o l . i n ml/kg and f o r E l e c t r o l y t e s i n mEq/kg.

X

R

Control (extreme values from 108 exp.)

0 0 0 0 0 0

NHCH CH=CH NHCH C=CH NHCH CH=CHCH NHCH CH CH CH NHiCHg^CHs 2

2

2

2

3

2

2

2

3

NHCHSCQHS

0

JQ^

NHCHs

0 S

NHCHSCQHS

NH CHg CH CH CH

NHCIfeC H5 NHd^CEfeCH CHQ NHCHgCeHs OCH CH CH CH OCHgCglL^

2

2

2

e

2

2

2

2

3

+

K

+

Cl"

Vol.

Na

0.5 4.2

0.05 0.45

0.06 0.50

0.06 0.28

5 5 7 55 4 25

0.5 0.5 0.9 4.5 0.5 5-0

0.51 0.15 0.45 1.15 0.20 1.5

0.5 0.4 0.9 5.4 0.4 5-8

12

1.4

0.52

0.9

28

5-2

Ο.85

2.2

20 11 7 19 14 16

2.4 1.5 1.0 2.8 1.9 2.1

0.68

2.4 1.0 0.7 2.0 1.4 2.0

0.46

0.55 0.64

0.61 0.4o

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

NIELSEN

AND

FEIT

Aminobenzoic Acid Diuretics

23

staff of the Department of Pharmacology and to the Huntingdon Research Centre, Huntingdon, England, for the diuretic screening. Literature Cited 1. Presented i n part at the 174th National Meeting of the American Chemical Society, Division of Medicinal Chemistry, Chicago, I l l i n o i s , August-September, 1977. 2. F e i t , P. W., Bruun, H. and Nielsen, C. Κ., J. Med. Chem. (1970), 13, 1071. 3. F e i t , P. W., J. Med. Chem. (1971), 14, 432. 4. F e i t , P. W. and Nielsen, O. B. T., J. Med. Chem. (1972), 15, 79. 5. F e i t , P. W., Nielsen, O. Β. T. and Rastrup-Andersen, N., J. Med. Chem. (1973), 16, 127. 6. Nielsen, O. B. T., Nielsen, C. K. and F e i t , P. W., J. Med. Chem. (1973), 16, 7. F e i t , P. W., Nielsen Chem. (1974), 17, 572. 8. Nielsen, O. B. T., Bruun, H . , Bretting, C. and F e i t , P. W., J. Med. Chem. (1975), 18, 41. 9. F e i t , P. W. and Nielsen, O. B. T., J. Med. Chem. (1976), 19, 402. 10. F e i t , P. W. and Nielsen, O. B. T., J. Med. Chem. (1977), 20, 1687. 11. Sprague, J. M. i n "Topics in Medicinal Chemistry," V o l . II, pp. 22-24, Rabinowitz, J. L . and Myerson, R. M . , E d . , Wiley, New York, Ν. Υ . , 1968. 12. F e i t , P. W., Nielsen, O. Β. T. and Bruun, H . , J. Med. Chem. (1972), 15, 437. 13. Østergaard, E . H . , Magnussen, M. P . , Nielsen, C. K . , Eilertsen, E . and Frey, H . - H . , Arzneim. Forsch. (1972), 22, 66. 14. Postgrad. Med. J. (1975), 51, Suppl. 6, "Bumetanide," Hoffbrand, Β. I . and Jones, G . , Ed. 15. F e i t , P. W. and Bruun, H . , U.S. Patent 3,864,385 (1973). 16. F e i t , P. W., Nielsen, O. B. T., Bretting, C. and Bruun, H . , U.S. Patent 4,082,851 (1978). RECEIVED

August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3 4-(3-Sulfamoylphenyl)thiazolidin-4-ols A Novel Class of Sulfonamide Compounds with Salidiuretic Activity H.-J. LANG, Β. KNABE, R. MUSCHAWECK, M. HROPOT, and E . LINDNER Hoechst Aktiengesellschaft, Postfach 80 03 20, D-6230 Frankfurt/M. 80, West Germany

Along with other developments, the history of pharmaceutical research in our laboratories shows a close connection with the history of diuretics. A few years after the discovery i n 1919 that antibiotic mercury compounds display diuretic effects (1, 2), investigators at Hoechst developed Salyrgan as the reference compound of the "Mercurials" (3). About 30 years ago, these organometallic drugs were displaced by sulfonamide derivatives which produced long acting diuresis (4, 5, 6). In 1959, research­ ers at Hoechst discovered the f i r s t potent short acting high­ -ceiling diuretic with a benzenesulfonamide structure, furosemide (7). Another interesting development in the f i e l d of high-ceiling diuretics was the synthesis of the acylphenoxyacetic acid deriva­ tive, ethacrynic acid (8). Another significant contribution to the high-ceiling diuretics which emanated from research in our laboratories was the development of piretanide (Hoe 118) during the last few years (9). The present report w i l l show the importance we attach to the long acting s a l i d i u r e t i c s . When we f i r s t began our research into long acting diuretics, only the sulfonamide derivatives were known. At that time all commercial diuretics with a thiadiazidel i k e s a l i d i u r e t i c action could be derived from two general formu­ las, i.e., either from 3-sulfamoylsulfonamides (Structure 1) or from 3-sulfamoylbenzamides (Structure 2). Chlorthalidone (Compound 3) was an exception to this generalization (Scheme I ) . Scheme I

3-Sulfamoylsulfonamides

2 3-Sulfamoylbenzamides

3 Chlorthalidone

0-8412-0464-0/78/47-083-024$05.00/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3.

LANG

ET AL.

4^3-Sulfamoylphenyl)thiazolindin-4'Ols

25

As very l i t t l e was known about the s t r u c t u r e - a c t i v i t y r e l a t i o n ­ ships of c h l o r t h a l i d o n e (10), we were i n t e r e s t e d i n e l u c i d a t i n g the s t r u c t u r a l features which are important f o r the s a l i d i u r e t i c a c t i v i t y . That i s , what i s the s i g n i f i c a n c e of the fused a r y l moiety, the carbamoyl moiety, the f i v e membered r i n g , and the N-C-OH moiety with respect to the s a l i d i u r e t i c a c t i v i t y ? Compounds 4a and 4b (Scheme I I ) show c h l o r t h a l i d o n e and i t s open chain tautomeric form (10). Scheme I I

To answer our f i r s t question as t o the importance o f the fused a r y l moiety, we decided to prepare dearyl compounds (Scheme I I I ) from α-haloacetophenones ( S t r u c t u r e 5) and b i f u n c t i o n a l molecules ( S t r u c t u r e 6 ) , where A represents the more n u c l e o p h i l i c p o r t i o n o f t h i s molecule. The B-H p o r t i o n o f S t r u c t u r e 7b i s then f r e e t o c y c l i z e with the ketone carbonyl group g i v i n g r i n g form ( S t r u c t u r e 7a) which would e x i s t i n tautomeric e q u i l i b r i u m with the open chain form. As i n d i c a t e d at the bottom, many such b i f u n c t i o n a l molecules ( S t r u c t u r e 6) were i n v e s t i g a t e d and many o f the r e s u l ­ tant compounds d i s p l a y s a l i d i u r e t i c e f f e c t s . Scheme I I I

Thioureas ( S t r u c t u r e 9) were used as the b i f u n c t i o n a l mole­ cules r e a c t i n g as expected (11, 12) with the ot-haloacetophenone ( S t r u c t u r e 8) to give isothiouronium s a l t s ( S t r u c t u r e 10b) (Scheme IV). These compounds undergo c y c l o t a u t o m e r i z a t i o n t o give t h i a z o l i d i n o l s a l t s ( S t r u c t u r e 10a). Treatment of these s a l t s with weak bases such as t r i e t h y l a m i n e or sodium bicarbonate a f f o r d s the f r e e bases ( S t r u c t u r e 11).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

26

AGENTS

Scheme IV

CI. X e.g., C l , Br 1

ΗΝ-"^-ΝΗ

R,

R

2

2 H

i H

3~~4β 7~

R

2

R

H

(a)

Base +HX

-HX

11 These thiazolidinylbenzenesulfonamides ( S t r u c t u r e s 10 and 11) were found to be potent s a l i d i u r e t i c agents and extensive m o d i f i c a t i o n s were performed to e l u c i d a t e f u r t h e r s t r u c t u r e - a c t i v i t y r e l a ­ t i o n s h i p s . The f o l l o w i n g comments with respect t o s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s p e r t a i n t o s t u d i e s with r a t s . S t r u c t u r e 12a o f Scheme V shows where m o d i f i c a t i o n s may be made.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3.

LANG ET

AL.

4-(3-Sulfarmylphenyl)thiazoUndin'4-oh

27

Scheme V

As mentioned above, there i s a tautomeric e q u i l i b r i u m between the t h i a z o l i d i n e and the open chain i s o t h i o u r e a forms ( S t r u c t u r e s 12a and 12b). T h i s e q u i l i b r i u m could be proved i n r e l a t e d s t r u c t u r a l systems by IR and NMR s p e c t r o s c o p i c s t u d i e s (JLX). The e q u i l i b r i u m i s e a s i l y observed i n the IR spectrum by examining the i n t e n s i t y of the carbonyl band near 1680 cm" , w h i c h i s caused only by the open chain form. When both R and R are r a t h e r bulky s u b s t i t uents, such as c y c l o h e x y l , c y c l o o c t y l and c y c l o d o d e c y l , there may be a carbonyl a b s o r p t i o n at 1680 cm" suggesting a greater^ 2 c o n t r i b u t i o n o f the open chain form. In the case where R and R are c y c l o d o d e c y l , there i s a marked r e d u c t i o n i n s a l i d i u r e t i c activity. T h i s reduced a c t i v i t y appears to be due more to the o v e r a l l i n c r e a s e d bulk o f the molecule than j u s t t o i t s e x i s t i n g i n the open chain form ( S t r u c t u r e 12b). Optimal s a l i d i u r e t i c a c t i v i t y i s a s s o c i a t e d with compounds where R i s a small subs t i t u e n t , such as methyl or ethyl, and R i s methyl, e t h y l or even a b u l k i e r s u b s t i t u e n t , such as c y c l o h e x y l , c y c l o o c t y l , benzyl, e t c . Dehydration o f the t h i a z o l i d i n e s to t h i a z o l i n e s ( S t r u c t u r e 13) i s r e a d i l y achieved by b o i l i n g with a c e t i c a c i d . These compounds show reduced s a l i d i u r e t i c a c t i v i t y when compared to the corresponding t h i a z o l i d i n e s ( S t r u c t u r e 12). p

Replacement o f one or both sulfamoyl hydrogen atoms produces a decrease or a l o s s of s a l i d i u r e t i c a c t i v i t y i n most of the known sulfamoyl d i u r e t i c s . We were q u i t e s u r p r i s e d to observe almost no decrease i n s a l i d i u r e t i c a c t i v i t y when one o f the sulfamoyl hydrogen atoms i n formula 10 or 11 was r e p l a c e d with a l k y l groups, f o r example, with methyl t o n-hexyl groups.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

28

AGENTS

Furthermore, almost no decrease i n activity,.was observed by s i m i l a r replacement o f one hydrogen atom (R or R ) o f formula 12 with phenyl, carbamoyl, a c e t y l o r a r a l k y l groups, such as benzyl or a s u b s t i t u t e d benzyl group. Good s a l i d i u r e t i c a c t i v i t y but a s l i g h t l y enhanced t o x i c i t y was observed when both hydrogen atoms o f the sulfamoyl group were replaced, f o r example, with methyls or with methyl and benzyl groups. At t h i s p o i n t we a l s o t r i e d to answer two other questions with respect to s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s . F i r s t , i s the sulfamoyl group necessary f o r s a l i d i u r e t i c a c t i v i t y ? A literature survey revealed compounds o f general s t r u c t u r e 14 where X or Y are not a sulfamoyl group were reported by Manning and Houlihan (12).

14

A s a l i d i u r e t i c a c t i v i t y i s a s s o c i a t e d with these compounds; however, compounds 12a c o n t a i n i n g a sulfamoyl group are much more potent as s a l i d i u r e t i c agents. The second question was whether or not the c h l o r i n e atom and sulfamoyl group could be interchanged. The isomer 15 of t i z o l e m i d e (Compound 16, Hoe 740) was synthesized and was found to be almost devoid o f s a l i d i u r e t i c a c t i v i t y .

15

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3. LANG ET AL.

4^3-Sulfamoylphenyl)thiazolindin-4-oh

29

Optimal a c t i v i t y appears to be a s s o c i a t e d with the u n s u b s t i tuted sulfamoyl group, so t i z o l e m i d e was s e l e c t e d f o r f u r t h e r investigations.

The hydrochloride s a l t substance which i s moderately s o l u b l e i n water. Most other sulfonamide d i u r e t i c s have an a c i d i c o r n e u t r a l f u n c t i o n a t the p o s i t i o n meta t o the sulfamoyl group. I n c o n t r a s t , t i z o l e m i d e bears a b a s i c s u b s t i t u e n t a t t h i s p o s i t i o n . Tizolemide was compared with h y d r o c h l o r o t h i a z i d e and c h l o r t h a l i d o n e i n v a r i o u s pharmacological assays. On the f o l l o w i n g f i g u r e s the a b b r e v i a t i o n s Hoe 740, HCT and Ch-on appear t o i n d i cate these agents. ION

EXCRETION IN RATS

Figure 1 shows the dose-response curves i n r a t s a f t e r o r a l a d m i n i s t r a t i o n o f t i z o l e m i d e , h y d r o c h l o r o t h i a z i d e and c h l o r t h a l i d o n e . Only sodium e x c r e t i o n i s shown, s i n c e e x c r e t i o n o f c h l o r i d e and water f o l l o w i n a p a r a l l e l manner. The curves are g e n e r a l l y f l a t over a dose range o f 0.25 t o 32 mg/kg which i s t y p i c a l f o r t h i a z i d e - l i k e d i u r e t i c s . However, a t higher dosages, Hoe 740 exerts an a d d i t i o n a l e f f e c t on the e x c r e t i o n o f sodium, c h l o r i d e and water. Micropuncture s t u d i e s i n r a t s revealed that the most i n t e n s i v e i n h i b i t i o n o f sodium-reabsorption by Hoe 740 i s l o c a t e d i n the d i s t a l tubule. A s l i g h t a c t i v i t y was a l s o observed i n the loop o f Henle which might account f o r the h i g h - c e i l i n g behavior of Hoe 740. ION

EXCRETION IN DOGS

Figure 2 shows the dose-response curves with respect t o the sodium and c h l o r i d e e x c r e t i o n i n dogs a f t e r o r a l a d m i n i s t r a t i o n o f t i z o l e m i d e , h y d r o c h l o r o t h i a z i d e and c h l o r t h a l i d o n e . I n c o n t r a s t to the previous r e s u l t s with r a t s , these agents are much more potent i n dogs. The curves are n e a r l y i d e n t i c a l . There was no s i g n i f i c a n t d i f f e r e n c e between these agents i n dogs over the dose range i n v e s t i g a t e d .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

30

Figure 1. Sodium excretion, rat, 5 hr

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978. Figure 2. Sodium and chloride excretion, dog, 6 hr

-

Η-

co

ΣΓ

ο

s* •κ

Ι

δ-

η

sr

•s

3 ο

ι

I

DIURETIC AGENTS

32

THE

ION

RATIO

I t i s thought that a good d i u r e t i c agent should cause l i t t l e or no enhanced e x c r e t i o n of potassium. I t has been suggested t h a t there i s i n many cases a c l o s e connection between the bicarbonate e x c r e t i o n and the e x c r e t i o n o f potassium a f t e r treatment with d i u r e t i c s (13, 14). F i g u r e 3 shows the r a t i o of C l " / N a + K i n dogs f o r Hoe 740, h y d r o c h l o r o t h i a z i d e and c h l o r t h a l i d o n e , produced i n the previous experiment ( F i g u r e 2) over the s a l u r e t i c a c t i v e dose range. In the t h e o r e t i c a l case where no other ions are excreted, t h i s r a t i o would be equal to 1. I f bicarbonate i s a l s o excreted, the r a t i o i s l e s s than 1. F i g u r e 3 shows a higher C l " / N a + K r a t i o f o r Hoe 740 than f o r h y d r o c h l o r o t h i a z i d e and c h l o r t h a l i d o n e . T h i s observation suggests that Hoe 740 would induce a lower e x c r e t i o n of potassium than c h l o r t h a l i d o n e +

+

+

+

SODIUM AND

POTASSIUM EXCRETIO

F i g u r e 4 shows the c o r r e l a t i o n between sodium and potassium e x c r e t i o n i n dogs a f t e r i . v . a d m i n i s t r a t i o n of 12.5 mg/kg/h of c h l o r t h a l i d o n e and Hoe 740. In comparison to the c o n t r o l group, there was a s i g n i f i c a n t i n c r e a s e i n sodium e x c r e t i o n a f t e r admini s t r a t i o n of both compounds. C h l o r t h a l i d o n e a l s o causes a s i g n i f i c a n t i n c r e a s e i n potassium e x c r e t i o n , while potassium excret i o n a f t e r Hoe 740 i s not s i g n i f i c a n t l y increased i n comparison t o the c o n t r o l group. T h i s o b s e r v a t i o n i s presumably c o n s i s t e n t with the higher C l " / N a + K r a t i o mentioned above f o r Hoe 740. +

+

ANTIHYPERTENSIVE EFFECTS F i g u r e 5 shows the a n t i h y p e r t e n s i v e e f f e c t s i n r a t s t r e a t e d with g l y c y r r h e t i n i c a c i d a f t e r a d a i l y a d m i n i s t r a t i o n of 50 mg/kg (p.o.) of Hoe 740, h y d r o c h l o r o t h i a z i d e and c h l o r t h a l i d o n e . The g l y c y r r h e t i n i c r a t i s a modified DOCA r a t , as developed by Prof. Dr. Lindner (Hoechst AG) by use o f g l y c y r r h e t i n i c a c i d i n p l a c e of l l - d e s o x y c o r t i c o s t e r o n - 2 1 - a c e t a t e . The high blood pressure i s produced a f t e r a c o n t r o l p e r i o d by treatment with T y r o d e - s o l u t i o n as the d r i n k i n g water and g l y c y r r h e t i n i c a c i d (10 mg/kg d a i l y ) . When a constant hypertension was achieved, the s a l i d i u r e t i c agent was administered while treatment with g l y c y r r h e t i n i c a c i d was continued. The blood pressure was s i g n i f i c a n t l y decreased a f t e r Hoe 740, as w e l l as, a f t e r treatment with the other two agents. When s a l i d i u r e t i c treatment was withdrawn, the blood pressure i n c r e a s e d again but the previous hypertensive l e v e l was not achieved. SERUM URIC ACID CONCENTRATIONS Subchronic s t u d i e s of the e f f e c t s o f Hoe 740 on serum u r i c a c i d concentrations have a l s o been performed. Serum u r i c a c i d

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3. LANG ET AL.

4^3-Sulfamoylphenyl)thiazolindin-4-ols

Figure 3.

Figure 4.

Cl/Na

+

+ K , dog +

Corrélation between Ήα* and Κ* excretion, dog

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

33

DIURETIC AGENTS

34

ι 5

Con trol

1 10

1 15

1 20

1 25

1 30

|

days

Glycyrrhetinic acid

Figure 5. Antihypertensive effect in glycyrrhetinic rat

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3.

LANG ET A L .

4-(3-Sulfamoylphenyl)thiazolindin-4-oh

Π

4

2

35

Ά

5 1

MOI

1251

1501

l10l

Dose, mg/kg

Figure 6.

150

25

50

11001

—

Effect of Hoe 740 on UA-serum concentration in comparison with chlorthalidone and hydrochlorothiazide in oxonic rat

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

36

DIURETIC AGENTS

concentrations have also been performed. Serum uric acid levels in dogs during 30 days treatment with 2.5, 8 and 25 mg/kg of Hoe 740 were not influenced; however, these levels were decreased in normal rats during the same length of treatment after oral administration of Hoe 740 at 4, 40 and 400 mg/kg. The effect of Hoe 740 on serum uric acid was also studied in the oxonic rat (15, 16). Most mammals with the exception of some primates and humans convert uric acid into allantoin by means of the enzyme uricase. Allantoin is readily excreted in the urine because of i t s water s o l u b i l i t y and i t s poor reabsorption in the kidney. Treatment of rats with the potassium salt of oxonic acid inhibits uricase and results in elevated serum uric acid levels. These hyperuricemic rats are treated once a day for three days with the test substance as well as oxonic acid. At the end of the test period, the animals sacrificed d level f uri acid are determined. Figur rat. In this test, chlorthalidon produce y sligh increase in serum uric acid levels. Hydrochlorothiazide in higher doses produced a significant decrease in serum uric acid levels. In contrast, at low doses, Hoe 740 displayed an antihyperuricemic a c t i v i t y , whereas at doses higher than 10 mg/kg, no significant difference from control animals was observed. Hoe 740 i s being investigated at the present time to elucidate the possible mechanism of this dose-related behavior. Of special importance is that decreased serum uric acid levels after 10 mg/kg of Hoe 740 (p.o.) could be demonstrated also in Cebus monkeys (cebus albifrons) whose metabolism of uric acid i s similar to man. The decrease of serum uric acid ô o n c e n t r a t i o n was significant (p<0.05) after Hoe 740 in comparison to the control group. Acknowledgments - We wish to thank Dr. Cragoe for the opportunity to present our work with these thiazolidines. We also wish to thank Dr. Lochelt, Dr. Schutz and Prof. Kramer for their subchronic studies in rats and dogs. Furthermore, I wish to thank my co-workers, Miss "Ansuhn, Mrs. Fischer and Mr. Ahlborn. I wish to thank Dr. Lawrence Martin for his assistance in the English translation. Literature Cited 1. Saxl, P. and H e i l i g , R., Wien. K l i n . Wschr. (1920), 33, 943. 2. Vogl, Α . , Amer. Heart J. (1950), 39, 881. 3. Hoechst, DRP. (1925) 423031; Friedlander 15, 1608. 4. American Cyanamid, U.S. Pat. 2554816 (1951). 5. Novello, F . C. and Sprague, J. M . , J. Amer. Chem. Soc. (1957), 79, 2028. 6. Ciba, B r i t . Pat. (1960), 847064. 7. Sturm, K . , Siedel, W., Weyer, R. and Ruschig, H . , Ber. Deut. Chem. G e s . (1966), 99, 328. 8. Schultz, Ε. Μ., Cragoe, E . J., Jr., Bicking, J. B . , Bolhofer, W. Α . , Sprague, J. M . , J. Med. Pharm. Chem. (1962), 5, 660. 9. Merkel, W., Bormann, D . , Mania, D . , Muschaweck, R. and

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

3. LANG ET AL.

4-(3-Sulfamoylphenyl)thiazolindin-4-ols

37

Hropot, M . , Eur. J. Med. Chem.-Chim. Ther. (1976), 11, 399. 10. Graf, W., Girod, E., Schmid, E . and S t o l l , W. G . , Helv. Chim. Acta (1959), 42, 1085. 11. Sharpe, C. J., Shadbolt, R. S., Ashford, A. and Ross, J. W., J. Med. Chem. (1971), 14, 977. 12. Houlihan, W. J. and Manning, R. E., U.S. P a t . . 3,671,533. 13. Muschaweck, R . , "Atti del Symposium Internazionale sul Potassio in Biologia ed i n Medicina," Siena, May 22-23 (1965). 14. Meng, K. and Loew, D . , "Diuretika: Chemie, Pharmakologie, Therapie," 184, Georg Thieme Verlag, Stuttgart (1974). 15. Musil, J. and Sandow, J., "Amino Acid Transport and Uric Acid Transport," Eds. Silbernagl, S., Lang, F . and Greger, R . , 227, Georg Thieme Verlag, Stuttgart (1975). 16. Musil, J., "Second International Symposium on Purine Metabolism i n Man," Eds Mathia M Muller Erich Kaise d J. Edwin Seegmiller, 179, RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

4 Sulfonamide Diuretics L. H . WERNER, E . HABICHT, and J. ZERGENYI Research Department, Pharmaceuticals Division, CIBA-GEIGY Corp., Summit, NJ 07901 and Research Department, Pharmaceuticals Division, CIBA-GEIGY Ltd., Basle, Switzerland

More than twenty the thiazide diuretics f i e l d have occurred. The discovery of the thiazide diuretics i n 1957-58 was the beginning of a new era in the treatment of edema and hypertension. Prior to the introduction of the thiazide diuretics, one had to rely on the mercurial diuretics and later, on the carbonic anhydrase inhibitors with their well known drawbacks. The carbonic anhydrase inhibitors acted mainly i n the proximal tubule leading to increased urinary excretion of Na , Κ , and HCO and, as a consequence, to metabolic acidosis. At the time the carbonic anhydrase inhibitors were being developed, it was, however, the firm conviction of Drs. Beyer (1) and Baer of Merck Sharp and Dohme that a sulfonamide diuretic could be found that was saluretic, i.e., that increased urinary Na and Cl excretion i n equivalent quantities and therefore would not produce metabolic acidosis, if i t worked at the appropriate site along the nephron. This working hypothesis eventually led to chlorothiazide. It is now known that, depending on the site of action in the nephron, different diuretic profiles can be obtained, ranging from the carbonic anhydrase inhibitors to the thiazides and high-ceiling diuretics as is shown in Figure 1 (2, 3). Chemical structures, representing different types of diuretics have varied to a considerable extent. The structures of these various classes of diuretics are exemplified by the carbonic anhydrase inhibitors, acetazolamide (4) and dichlorphenamide (5), the thiazide saluretic, chloro­ thiazide (6), the high-ceiling diuretics, furosemide (7), ethacrynic acid (8) and bumetanide (9), the uricosuric diuretic, t i e n i l i c acid (10), and the more recent compounds, muzolimine (11) and MK-447 (12), the last two representing compounds without a sulfonamide or carboxy group (Figure 2). Some of the work that was carried out in our laboratories in the area of sulfonamide diuretics w i l l be presented. The sulfonamide diuretics currently in use are effective and safe; +

+

-

3

+

-

0-8412-0464-0/78/47-083-038$05.00/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WERNER ET AL.

Figure 1.

Sulfonamide Diuretics

Representation of sites of action of diuretics in the nephron

TABLE I D i u r e t i c Screen Reference Compounds

Unanesthetized Dog

Hydrochlorothiazide 5.0 mg/kg p.o.

1300 yEq Na/kg/210 min.

Furosemide 5./0 mg/kg p.o.

5200 yEq Na/kg/210 min.

Bumetanide 0.3 mg/kg p.o.

5700 yEq Na/kg/210 min.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

40

Cl Cl

Cl

S0 NH

H N0 S 2

2

2

S ' 0

H N0 S

a

2

2

2

chlorothiazide

dichlorphenamide

^3 H N0 S 2

>

2

C H

COOH

2

CH =Ç-C0—fi 2

3

\ — 0CH C00H 2

\-=/

ethacrynic acid

furosemide

Cl

Οχ

Cl

J-4 0CH C00H 2

H N0 S ^ ^ s ^ ^ C O O H 2

2

tienilic

bumetanide

_ /

N

H

acid

2

CH NH 2

CH

Cl

a

Cl muzolimine Figure 2.

MK 447 Various classes of diuretics

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2

4. WERNER ET AL.

Sulfonamide Diuretics

41

there a r e , however, three u n d e s i r a b l e s i d e e f f e c t s a s s o c i a t e d with the chronic a d m i n i s t r a t i o n of these compounds: (1) potassium d e p l e t i o n , (2) e l e v a t i o n o f serum u r i c a c i d and (3) hyperglycemia. In p a t i e n t s with a normal f a s t i n g blood sugar, i t would appear that the r i s k o f p r e c i p i t a t i n g diabetes i s s m a l l . In p a t i e n t s who already have diabetes, the chance of d i s t u r b i n g the c o n t r o l o f blood sugar i s s u b s t a n t i a l . We hoped that our work would provide compounds e i t h e r with l e s s e f f e c t on potassium e x c r e t i o n o r on blood glucose l e v e l s . The compounds prepared were t e s t e d i n r a t s and dogs; however, only r e s u l t s i n dogs are reported as an i n d i c a t i o n o f the r e l a t i v e potency o f these compounds. Table I shows the values obtained with three standard sulfonamide d i u r e t i c s i n our d i u r e t i c screening using unanesthetized dogs. The values shown i n F i g u r e s 3 to 7 a l s o represent yEq Na/kg/210 min In our f i r s t approac compounds derived from intermediate active diuretics. I n i t i a l l y , we s t u d i e d compounds derived from the 2-hydrazino-5-sulfamoylbenzoic a c i d (Compound 1) p r e v i o u s l y described by Sturm and co-workers (7) (Figure 3). The hydrazone (Compound 2) derived from phenylacetaldehyde, was only s l i g h t l y a c t i v e . Hydrazines such as compound 3 obtained by c a t a l y t i c r e d u c t i o n o f the corresponding hydrazone were moderately a c t i v e at 5 mg/kg i n the dog. On r e f l u x i n g i n d i l u t e HC1, the hydrazine (Compound 3) c y c l i z e d to the indazolone (Compound 5 ) , which was almost i n a c t i v e . F i s c h e r i n d o l e r i n g c l o s u r e o f the hydrazone (Compound 2) i n a c e t i c a c i d y i e l d e d the i n d o l e (Compound 4) which e x h i b i t e d d i u r e t i c a c t i v i t y a t 50 mg/kg. A l l compounds were administered orally. Reaction o f the 2-hydrazino-5-sulfamoylbenzoic a c i d (Compound 1) with cyclohexanone (Figure 3) followed by c y c l i z a t i o n y i e l d e d the t e t r a h y d r o c a r b a z o l e (Compound 6) which was subsequently reduced c a t a l y t i c a l l y i n t r i f l u o r o a c e t i c a c i d with platinum to the hexahydrocarbazole (Compound 7 ) . Both compounds 6 and 7 had moderate d i u r e t i c a c t i v i t y a t 100 mg and 50 mg/kg i n the dog. Another approach s t a r t e d with 2 , 4 - d i c h l o r o - 3 - n i t r o - 5 sulfamoylbenzoic a c i d (Compound 8, F i g u r e 4 ) . Treatment o f compound 8 with sodium hydroxide y i e l d e d the s a l i c y l i c a c i d d e r i v a t i v e (Compound 9) (13). Reduction of the n i t r o group i n compound 9 followed by f u s i o n with b u t y r i c anhydride o r benzoic anhydride gave the benzoxazoles (Compounds 10a and 10b, respect i v e l y ) . Only the 2-phenyl d e r i v a t i v e (Compound 10b) showed a p p r e c i a b l e a c t i v i t y at' 20 mg/kg i n the dog. S i m i l a r l y , the benzimidazole (Compound 12, F i g u r e 4) was prepared. I t was i n a c t i v e . S t a r t i n g with the 3-amino-4a n i l i n o - 5 - s u l f a m o y l b e n z o i c a c i d (Compounds 13a (9) and 13b), two other benzimidazoles (Compounds 14a and 14b, F i g u r e 4) were prepared, but were a l s o i n a c t i v e as d i u r e t i c s .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

42

CI H N0 S 2

2

H NO a

COOH

3700 y e q N a 100 mg/kg

Figure 3.

COOH

+

2400 \ieq Na+ 50 mg/kg

Compounds derived from the 2-hydrazino-5-sulfamoylbenzoic acid

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WERNER ET AL.

13

Sulfonamide Diuretics

a) R=H b) R=NH

a

43

14 a) Ri=R =H b) Ri=NH Ra-C H (n) 2

a

e

Figure 4.

7

Benzoxazole and benzimidazole derivatives

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

Figure 5. Tricyclic derivatives

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

4. WERNER ET AL.

Sulfonamide Diuretics

Figure 6. Phenoxazines and phenothiazines

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

45

DIURETIC AGENTS

46

CH 2CH 2R

(CH ) CH NH 2

2

2

CH =CHC0NH or CH =CH-CN 2

2

2

COOH

H N0 S 2

2

H N0 S

(COOH

27

a) R= C0NH b) R= CN

2

26

a) R=C1 b) R=OC H 6

s

2

2

2

28

2

34

35 ^ 6000 yeq Na 1.25 mg/kg

36

COOH

H N0 S

a) n=l< R-Cl, 0 C H b) n-2j

' V / 3500 yeq Na 1.25 mg/kg

^ 6000 yeq Na 1.25 mg/kg

Figure 7. Monocyclic aromatic sulfonamide derivatives

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

e

3

4. WERNER ET AL.

Sulfonamide Diuretics

47

T r i c y c l i c d e r i v a t i v e s , as shown i n Figure 5, were a l s o investigated. The r e a c t i o n o f o-aminophenol with 4-chloro-5c h l o r o s u l f o n y l - 3 - n i t r o - b e n z o i c a c i d (Compound 15) (14) was o f i n t e r e s t . Depending on the solvent, ethanol o r THF, s u l f o n y l a t i o n occurred on oxygen o r n i t r o g e n . Ring c l o s u r e o f the sulfonamide intermediate i n aqueous NaHCO- y i e l d e d compound 16, whereas the s u l f o n y l o x y intermediate r i n g closed d i r e c t l y to y i e l d the isomeric t r i c y c l i c compound 17 (Figure 5 ) . The s t r u c t u r e o f compound 17 was confirmed by reduction o f the n i t r o group to the amine and r i n g closure to the imidazole d e r i v a t i v e (Compound 18) by r e f l u x i n g i n a c e t i c anhydride. S e v e r a l d e r i v a t i v e s o f these two t r i c y c l i c compounds were prepared, f o r example, compounds 19 and 20, both were i n a c t i v e as d i u r e t i c s (Figure 5 ) . Reaction o f o-acetaminophenol i NaHCO s o l u t i o with the sulfamoylbenzoi N-acetyldiphenylamine d e r i v a t i v (Compoun ) yiel (Figure 6 ) . Whether t h i s compound a r i s e s by d i r e c t r e a c t i o n o f the h i g h l y r e a c t i v e chlorosulfamoylbenzoic a c i d (Compound 21) with the acetamino group o f the phenol or i s formed v i a a Smiles rearrangement o f an intermediate diphenyl ether has not been explored. The main r e a c t i o n product was the phenoxazine (Com­ pound 23)· S i m i l a r r i n g c l o s u r e r e a c t i o n s v i a a Smiles r e a r ­ rangement to phenoxazines and phenothiazines, e.g., compound 24 to compound 25, have been reviewed by Truce, e t a l (15). The phenoxazine (Compound 23) as well as the deacetylated d e r i v a t i v e had no d i u r e t i c a c t i v i t y . C e r t a i n monocyclic aromatic sulfonamide d e r i v a t i v e s were a l s o studied. S t a r t i n g with the 3-amino-5-sulfamoylbenzoic a c i d (Compound 26, F i g u r e 7) (9, 14), amino group was replaced by a propionamide o r p r o p i o n i t r i l e group under c o n d i t i o n s o f a Meerwein r e a c t i o n (Compound 16) using acrylamide or a c r y l o n i t r i l e , r e s p e c t i v e l y , to y i e l d compounds 27a and 27b. A Hoffmann degradation o f compound 27a gave the aminoethyl d e r i v a t i v e (Compound 28a). Reduction o f the n i t r i l e (Compound 27b) y i e l d e d the aminopropyl d e r i v a t i v e (Compound 28b, F i g u r e 7 ) . The N - a c e t y l d e r i v a t i v e (Compound 29) was i n a c t i v e . The e f f e c t of i n s e r t i n g a three carbon chain i n the 3 - p o s i t i o n o f the sulfamoylbenzoic a c i d moiety o f the bumetanide-type d i u r e t i c s was studied. Compound 30, which i s r e l a t e d to bumetanide, was only weakly a c t i v e , as was compound 31ι which d i f f e r s from furosemide only by a CHp group (Figure 7 ) . T h i s may be r e l a t e d to the increased b a s i c i t y o f the b e n z y l i c amine f u n c t i o n . F e i t and co-workers have shown that the aromatic amino group i n bumetanide can be replaced by an 0- o r S- ether linkage without a pronounced change i n d i u r e t i c potency; however, i n the a n t h r a n i l i c a c i d (furosemide) s e r i e s , the s t r u c t u r a l requirements are more s t r i n ­ gent. I t i s g e n e r a l l y accepted that s u b s t i t u t i o n on the s u l ­ fonamide n i t r o g e n o f sulfonamide d i u r e t i c s lowers the d i u r e t i c t

n

e

American Chemical Society Library 1155 16th St. N. W. In Diuretic Agents; Cragoe, E.; Washington, D. C. 20031

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

48 TABLE I I

32

a:

yEq/kg/4 hr Na K

Dose mg/kg p.o.

R NH

b:

NHCH

c:

NHCH C H

d:

NHC H

6

6

slight

5

3

+

5000

6.25

2

2

+

Ref.

935

(Furosemide)

activity

5

6.25

5

Control

2200

400

370

340

(18)

TABLE I I I

N H C H

„ sV r - ^ ^

R

^ o

^COOH

2

\ f

33

Dose mg/kg p.o.

It a:

p-Cl

b:

P-NH

c:

p-NH-CH

d:

p-N(CH )

e:

o-NH

5 5

2

3

3

2

Control

2

2-0

5

UEq/kg/4 hr Na

+

very s l i g h t

slight

+

Ref.

activity

5000

800

(18)

activity

inactive

5 2.5

K

5500

900

370

340

(18)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

4.

WERNER ET AL.

Sulfonamide Diuretics

49

Figure 8. Comparative dose response curves—furosemide vs. compound 32d—Νa* excretion in dogs

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 9.

Comparative dose response curves—furosemide vs. compound 32d—K* excretion in dogs Ω

α

WERNER ET AL.

4.

Sulfonamide Diuretics

51

a c t i v i t y . S i n c e we were not p r i m a r i l y l o o k i n g f o r enhanced potency but were i n t e r e s t e d i n t r y i n g to modify some of the other p r o p e r t i e s such as potassium d e p l e t i o n and e f f e c t s on blood glucose l e v e l s , we i n v e s t i g a t e d t h i s area. Compound 32a (Table I I ) i s furosemide, methylation o f the sulfamoyl n i t r o g e n as i n compound 32b d r a s t i c a l l y reduced the d i u r e t i c a c t i v i t y , the N-benzyl d e r i v a t i v e (Compound 32c) r e tained a modest degree of a c t i v i t y . S u r p r i s i n g l y , the N-phenyl d e r i v a t i v e (Compound 32d) was q u i t e n a t r i u r e t i c and appeared to have very l i t t l e e f f e c t on potassium e x c r e t i o n as shown i n Table II. This observation was f u r t h e r explored by i n t r o d u c i n g a s u b s t i t u e n t on the phenyl r i n g attached to the sulfamoyl group of compound 32a. The p-chlorophenyl d e r i v a t i v e (Compound 33a, Table III) was only weakly a c t i v e however th p-aminophenyl d e r i v a t i v e (Compound 33b) prove a t i o n of the amino group compound greatly reduced the a c t i v i t y ; the 4-dimethylaminophenylsulfonamide (Compound 33d) was a c t u a l l y i n a c t i v e a t the 5 mg/kg dose (Table III). The o-aminophenylsulfonamide d e r i v a t i v e (Compound 33e) was more a c t i v e on a weight b a s i s than the p-aminophenyl d e r i v a t i v e (Compound 33b). Unfortunately, potassium e x c r e t i o n was a l s o elevated. The f i n d i n g that a para- or ortho-aminophenyl group can enhance a c t i v i t y was a l s o a p p l i e d to the 3-amino-5-sulfamoylbenzoic a c i d s e r i e s (Figure 7 ) . Compound 34 had moderate d i u r e t i c a c t i v i t y at 1.25 mg/kg p.o. i n the dog. Both compounds 35 and 36 were a c t i v e d i u r e t i c s (1£, 20). Compound 36 was of somewhat g r e a t e r i n t e r e s t and was a c t i v e i n a dose r e l a t e d sense over a range of 0.02 mg to 1 mg/kg. Thus, a number of compounds of p o t e n t i a l i n t e r e s t had been found and considered worthy of f u r t h e r study. Compound 32d (Table I I ) was of i n t e r e s t because i t appeared to have very l i t t l e e f f e c t on potassium e x c r e t i o n . I t was an a c t i v e d i u r e t i c i n dogs and reached a n a t r i u r e t i c c e i l i n g at a dose of approximately 6 mg/kg. Osmolar clearance increased two-to t h r e e - f o l d , while r e a b s o r p t i o n o f solute f r e e water remained p o s i t i v e i n the dog. F i g u r e 8 shows the dose response curve f o r sodium e x c r e t i o n i n dogs. At higher dosages, sodium e x c r e t i o n l e v e l e d o f f f o r compound 32d, whereas i t continued upward with furosemide. Figure 9 shows the e f f e c t on potassium e x c r e t i o n , which was p r a c t i c a l l y unaffected by higher doses. C l i n i c a l t r i a l s i n normal volunteers, however, f a i l e d to produce any d i u r e t i c e f f e c t s . Compound 33b was a l s o studied i n greater d e t a i l . The d i u r e t i c e f f e c t s i n the dog resembled those obtained with f u r o semide, except that at doses above 10 mg/kg, the e x c r e t i o n of Na , C l " , K and water decreased somewhat (Figure 10). During peak d i u r e s i s , the urine was almost i s o t o n i c . Renal plasma flow +

+

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

52

DIURETIC AGENTS

Figure 11. Diuretic effects of compound 33e and furosemide in dogs—excretion per dog per 6 hr

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WERNER ET AL.

Sulfonamide Diuretics

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

54

DIURETIC AGENTS

and glomerular f i l t r a t i o n rate were only slightly affected. In normal volunteers, doses of 40 to 120 mg produced a rapid onset of diuresis which persisted for 5 to 6 hours, similar to that of furosemide. The natriuretic effect reached a ceiling at the 80 to 120 mg dose and was about equal to 40 mg of furosemide. Compound 33e was a somewhat more potent diuretic in dogs than furosemide. The onset of action was prompt, reaching a peak after 90 minutes and subsiding aft^r 5 to 6 hours. The dose re­ sponse curves for urine volume, Na , Κ and Cl"" are shown in Figure 11. In normal volunteers, contrary to the results obtained in animal experiments, compound 33e was not a high-ceiling diuretic. An activity plateau was reached at 80-120 mg, two to three times the threshold dosage (Figure 12). Its natriuretic effect at this level was less than that induced by 100 mg of hydrochlorothiazide Interestingly, this compoun excretion, but rather a persisting hours. This type of diuretic response may be desirable in the treatment of hypertension. Unfortunately, none of the compounds described were potassium-sparing nor did they have appreciably less effect on glucose tolerance; however, no uricosuric effect was observed. This brief overview of some of our work on sulfonamide diuretics illustrates one of the problems we encounter in medicinal chem­ i s t r y ; namely, that encouraging biological data in animals do not always translate into equally favorable c l i n i c a l results. Acknowledgments - We wish to thank Dr. O. Bîîch, Dr. P. R. Hedwall and Dr. J. Kraetz, Basle, and Dr. M. J. Antonaccio, Dr. W. E. Barrett and Mr. R. Rutledge, Summit, for the pharma cological data and Dr. P. R. Imhof, Basle, for the c l i n i c a l data. Literature Cited 1. Beyer, Κ. Η., Jr., Perspectives in Biology and Med. (1976), 19, 500. 2. Jacobson, H. R. and Kokko, J. P., Ann. Rev. Pharmacol. Toxicol. (1976), 16, 201. 3. Anderton, J. L . and Kincaid-Smith, P., Drugs (1971), 1, 54. 4. Roblin, R. O., Jr., and Clapp, J. W., J. Am. Chem. Soc. (1950), 72, 4890. 5. Beyer, Κ. H. and Baer, J. Ε . , Pharmacol. Rev. (1961), 13, 517. 6. Novello, F. C . , B e l l , S. C . , Abrams, E. L . Α . , Ziegler, C. and Sprague, J. M . , J. Org. Chem. (1960), 25, 965. 7. Sturm, K . , Siedel, W., Weyer, R. and Ruschig, Η . , Chem. Ber. (1966), 99, 329. 8. Schultz, E. M . , Cragoe, E. J., Jr., Bicking, J. Β . , Bolhofer, W. A. and Sprague, J. M . , J. Med. Chem. (1962), 5, 660. 9. F e i t , P. W., J. Med. Chem. (1971), 14, 432. 10. T h u i l l i e r , G . , Laforest, J., Cariou, B. Bessin, P., Bonnet, J. and T h u i l l i e r , J., Eur. J. Med. Chem. (1974), 9, 625. 11. M ö l l e r , Ε . , Horstmann, Η . , Meng, K. and Loew, D . , Experientia +

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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55

(1977), 33, 382. 12. Affrime, Μ. Β., Lowenthal, D. T., Onesti, G. Busby, P . , Swartz, C. and L e i , Β., C l i n . Pharmacol. Ther. (1977), 21, 97. 13. Liebenow, W., Canadian Patent 952,536 (1974), Chem. Abstr. (1975), 82, P170,430. 14. Feit, P. W., Bruun, H. and Nielsen, C. K . , J. Med Chem. (1970), 13, 1071. 15. Truce, W. E., Kreider, E . M. and Brand, W. W., "Organic Reactions", V o l . 18, p. 99, E d . , W. G. Dauben, John Wiley & Sons, Inc., New York, 1970. 16. Müller, Ε . , Angew. Chem. (1949), 61, 179. 17. Feit, P. W., Tvaermose-Nielsen, O. B. and Bruun, H., J. Med. Chem. (1974), 17, 572. 18. Werner, L . H., U.S. Patent 3,812,104 (1974); Chem. Abstr. (1969), 70, P67908r. 19. Werner, L . H., U.S (1976), 84, P105217p. 20. Werner, L . H., U.S. Patent 3,939,267 (1976); Ger. Offen. 2,349,900 (1974). RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

5 Diuretic and Uricosuric Properties of Tienilic Acid (Ticrynafen) in Mice, Rats, and Anesthetized Beagle Dogs Antihypertensive Activity in SH Rats and Structure—Activity Relationship P. BESSIN, J. BONNET, M. F. MALIN, C. JACQUEMIN, N. D E BREZE, I. PELAS, L . DESGROUX, B. AGIER, and M. DUTARTE

Albert Rolland Center for Research and Pharmacology (CREPHAR), 4, Rue de la Division Leclerc, 91380 Chilly-Mazarin, France

In the last twenty discovered which exhibi These are (a) the phenoxyacetic acids, the most prominent member being ethacrynic acid (1,2, Figure 1) and (b) the sulfamoylbenzoic acids, the most potent being furosemide (3) and bumetanide (4,5). The latter compound differs from furosemide in several respects, the most obvious being that the 5-chloro substituent is replaced by phenoxy. These very potent natriuretic agents are sufficiently effective to satisfy the physician for use in the treatment of hypertension and cardiac diseases; however, they possess unwanted side effects which include potassium depletion, a diabetogenic propensity (6,7,8,9) and hyperuricemia (10-23). To solve the problem of potassium depletion, a search for new diuretic agents led to the discovery of compounds which block the tubular sodium-potassium exchange either directly, i.e., triamterene (24) and amiloride (25), or by blocking the action of aldosterone, i.e., spironolactone. In regard to diuretic-induced hyperuricemia, the discovery in 1967 of t i e n i l i c acid was a major contribution to diuretic therapy. This compound, (2% 3-dichloro-4-(2-thienylcarbonyl)phenoxy7acetic acid, is an aryloxyacetic acid analog of ethacrynic acid which exhibits diuretic and uricosuric activity in mice, rats and dogs (28), as well as in man (29,30,31). More recently, another aryloxyacetic acid, an indanone (32,33,3^,35), has been reported to possess potent diuretic and uricosuric activity in chimpanzees and i n man. The purpose of the studies which we w i l l describe was to evaluate the diuretic and uricosuric effects of t i e n i l i c acid in mice, rats and dogs and the antihypertensive properties in SH rats preliminary to c l i n i c a l t r i a l s . METHODS Diuretic and Uricosuric Activity in Mice (36) - I n i t i a l screening 0-8412-0464-0/78/47-083-056$07.25/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

5.

BESSIN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

ETHACRYNIC ACID

BENZIODARONE

Figure 1.

Compound structures

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

57

DIURETIC AGENTS

58

of d i u r e t i c agents were performed using groups o f 6 male Swiss mice, weighing 22 + 1 g, randomly s e l e c t e d and d i s t r i b u t e d i n p a i r s i n t o metabolism cages f o r 2 o r 4 hours. Food and water were withheld f o r 2 hours p r i o r to the experiments, u r i n e was c o l l e c t e d at 2 and 4 hours and the urine volumes and e l e c t r o l y t e s measured. A l l mice were administered 1 ml o f 0 . 9 Î s a l i n e o r a l l y and e i t h e r simultaneously t r e a t e d o r not ( c o n t r o l group) with d i u r e t i c agents. Test compounds ( t i e n i l i c a c i d , e t h a c r y n i c a c i d and furosemide) were d i s s o l v e d o r suspended i n the same 0 . 9 Ï s a l i n e and administered by gavage. Four groups r e c e i v e d t i e n i l i c a c i d o r a l l y a t doses o f 20, 50, 100 and 200 mg/kg and were compared to furosemide a t 20 mg/kg, e t h a c r y n i c a c i d a t 20 mg/kg and benziodarone (a potent n o n - d i u r e t i c u r i c o s u r i c agent) a t 100 mg/kg by the same route. F o r the g e n e r a l s c r e e n i n g o f d i u r e t i c and u r i c o s u r i c drugs, such tienili acid d it th compounds were administere comparison t o f u r o s e m i d Na and Κ analyses were determined u s i n g the flame photometer (Eppendorf), c h l o r i d e s by m i c r o a n a l y s i s (37) and u r i c a c i d by the enzymatic spectrophotometric technique (3SJ or by a colôrimetric method (.29). S t a t i s t i c a l a n a l y s i s was made by a p p l i c a t i o n o f Student's t t e s t w i t h simultaneous c a l c u l a t i o n o f means (M) and standard d e v i a t i o n o f the mean (S/VrT). +

T i e n i l i c Acid-Benziodarone and Furosemide-Benziodarone R e l a t i o n s h i p s i n Mice - Groups o f 6 mice weighing 22 + 1 g were admini s t e r e d simultaneously e i t h e r 100 mg/kg o r 200 mg/kg o f t i e n i l i c a c i d , o r 200 mg/kg o f benziodarone, o r 20 mg/kg o f furosemide o r a combination o f the same doses o f t i e n i l i c a c i d + benziodarone o r furosemide + benziodarone. A f t e r dosing, a l l animals were d i s t r i b u t e d by p a i r s i n t o metabolism cages f o r 2 hours. Urine samples were c o l l e c t e d f o r the determination o f e l e c t r o l y t e and u r i c a c i d e x c r e t i o n using the same a n a l y t i c a l methods as p r e v i ously d e s c r i b e d . The s t a t i s t i c a l s i g n i f i c a n c e was based on a comparison o f t i e n i l i c a c i d + benziodarone or furosemide + benziodarone versus t i e n i l i c a c i d alone, furosemide alone o r benziodarone alone (Student's t t e s t ) . D i u r e t i c A c t i v i t y i n Rats - Groups o f 3 animals weighing 400 + 5 g, deprived o f food and water o v e r n i g h t , were given 20 ml/kg o f water by i n t u b a t i o n on the day preceding the experiment. A f t e r dosing, the animals were placed i n metabolism cages f o r the c o l l e c t i o n o f a 5 hour sample o f u r i n e . D i u r e t i c drugs were administered as i n the previous experiment i n a volume o f 20 ml/kg of 0.9% s a l i n e a t the beginning o f the experiment, while a c o n t r o l group was administered only the s a l i n e s o l u t i o n . Statistical s i g n i f i c a n c e r e f e r s to a comparison between t r e a t e d and c o n t r o l r a t s (Student's t t e s t ) . Dose-response r e l a t i o n s h i p s are i l l u s t r a t e d i n F i g u r e 5 u s i n g the r e g r e s s i o n l i n e procedure.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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59

Phenol-Red Retention Test i n Rats (40,41) - Groups o f 5 randomized male r a t s , weighing 180 + 2 0 g, were given the t e s t compounds o r a l l y 30 minutes before the intravenous i n j e c t i o n o f 75 mg/kg o f phenol-red as a 1.5% s o l u t i o n i n 0.9% sodium c h l o r i d e . Heparini z e d blood samples were taken from the r e t r o - o r b i t a l plexus a t 15, 30, 45 and 60 minutes; c o l o r was developed by the a d d i t i o n o f 0.05 ml o f 0.1 M sodium hydroxide and read a t 540 my on the s p e c t r o c o l o r i m e t e r (Eppendorf). R e s u l t s were c a l c u l a t e d as percentage o f v a r i a t i o n i n r e l a t i o n to the c o n t r o l value. S t a t i s t i c a l s i g n i f icance was c a l c u l a t e d by comparison with t r e a t e d and c o n t r o l r a t s (Student's t t e s t ) . A n t i h y p e r t e n s i v e A c t i v i t y i n Spontaneous Hypertensive Rats (Wistar-Okamoto S t r a i n ) - The s t u d i e s were performed i n Wistar hypertensive male r a t s which were approximately 25 weeks o l d and randomly placed i n group pressure was determined Narco System) before treatment and a t 2, 5 and 24 hours a f t e r each d a i l y a d m i n i s t r a t i o n o f the drug o r the v e h i c l e , i n acute (1 day) and i n subacute experiments (8 days). In c h r o n i c s t u d i e s , the blood pressure was measured i n d i r e c t l y before and 1.5, 3·5, 6, 12 and 18 months a f t e r the beginning o f the experiment. In the l a s t study, t i e n i l i c a c i d was mixed with the d a i l y food a t a l e v e l c a l c u l a t e d to provide an o r a l intake o f 100 and 200 mg/kg. In the acute o r a l experiments, the r a t s (N = 6) were given 100, 200 and 400 mg/kg o f t i e n i l i c a c i d ; i n the subacute e x p e r i ments, the d a i l y o r a l dose was 200 mg/kg. S t a t i s t i c a l a n a l y s i s was determined by Tukey's m u l t i p l e comparisons procedure which permits the s t a t i s t i c a l c l a s s i f i c a t i o n o f means between themselves or compared to c o n t r o l data. In F i g u r e 17, s t a t i s t i c a l s i g n i f i cance i s i l l u s t r a t e d by the a d d i t i o n of a broken l i n e to the main s o l i d l i n e . Each p o i n t represents a mean value, and the v e r t i c a l bars i n d i c a t e the standard e r r o r o f the mean. In the chronic study, s t a t i s t i c a l s i g n i f i c a n c e was c a l c u l a t e d by Student's t p a i r e d t e s t . In a l l the experiments, the blood pressure was measured i n d i r e c t l y i n a c o n t r o l group o f SH r a t s under the same c o n d i t i o n s ^ I n a d d i t i o n , i n the c h r o n i c study, the t r e a t e d SH r a t s were compared to c o n t r o l Wistar normotensive r a t s . T r i g l y c e r i d e - L o w e r i n g E f f e c t i n Obese Rats ( F a t t y S t r a i n ) Measurement o f the t r i g l y c e r i d e - l o w e r i n g e f f e c t was c a r r i e d out i n groups o f 5 to 7 obese male r a t s ( F a t t y s t r a i n ) and compared to normal heterozygous r a t s as a f i r s t c o n t r o l group and to normal Wistar r a t s as a second c o n t r o l group. Obese r a t s were randomly d i s t r i b u t e d i n t o two groups o f 5 to 7 animals which were given e i t h e r 200 mg/kg o f t i e n i l i c a c i d as an o r a l d a i l y dose f o r 7 days or the v e h i c l e alone. The other c o n t r o l groups (heterozygous r a t s and normal Wistar r a t s ) were given only the f l u i d v e h i c l e . Blood samples were taken a t the end o f the

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

60

experiment from the r e t r o - o r b i t a l plexus i n order to determine plasma glucose, t o t a l l i p i d s , c h o l e s t e r o l and t r i g l y c e r i d e l e v e l s a f t e r d a i l y treatment with t i e n i l i c a c i d . Statistical signifi­ cance was c a l c u l a t e d i n comparison with t r e a t e d and c o n t r o l r a t s (Student's t t e s t ) . D i u r e t i c , u r i c o s u r i c and Clearance S t u d i e s i n Anesthetized Beagle Dogs under Hydropenic C o n d i t i o n s - The d i u r e t i c , u r i c o s u r i c and clearance s t u d i e s were performed i n beagle dogs weighing 10 t o 13 kg which had been f a s t e d overnight and then a n e s t h e t i z e d by i . v . i n j e c t i o n o f mebubarbital, 30 mg/kg. During the experiment, the dogs were i n t r a v e n o u s l y i n f u s e d a t a r a t e o f 2 ml/minute w i t h 0.6% s a l i n e , 0.08% PAH, 0.4% c r e a t i n i n e and mebubarbital, 5 mg/kg/hour u s i n g a constant r a t e i n f u s i o n pump. A f t e r m e s i a l laparotomy and c a t h e t e r i z a t i o n o f the u r e t e r s h stud s t a r t e d with control p e r i o d o f two hours. Bloo minute i n t e r v a l s . Then lysin i n j e c t e d i n t r a v e n o u s l y a t 5, 10 and 20 mg/kg. A group o f 4 t o 6 beagle dogs was used f o r each dose and f o r the c o n t r o l (placebo) e v a l u a t i o n . Sodium and potassium determinations were made u s i n g the Eppendorf Flame Photometer, c h l o r i d e by the c o l o r i m e t r i c micromethod (37) and urate by the enzymatic procedure (38). Other measurements and c a l c u l a t i o n s which were made are as f o l l o w s : PAH (42), c r e a t i n i n e (43), urea (44), pH, blood pressure, osm ( o s molar c l e a r a n c e ) a f t e r c c y o s c o p i c determination (using the f r e e z ­ i n g p o i n t d e p r e s s i o n ) , T-HpO ( f r e e water reabsorption) and Τ H 0 = osm - g (ml/min). The osm - Τ H O r e l a t i o n s h i p was studied u s i n g Τ HpO c o r r e c t e d f o r osmolar clearance, and c r e a t i n i n e c l e a r ­ ance as p r e v i o u s l y described (45): Τ Η 0 / osm. A l l the c l e a r 2

ρ

creat. ance s t u d i e s were determined using standard procedures. S t a t i s ­ t i c a l s i g n i f i c a n c e was c a l c u l a t e d as p r e v i o u s l y described by the a p p l i c a t i o n o f Tukey's t e s t (Section 5 ) . Data are i l l u s t r a t e d i n v a r i o u s f i g u r e s as the mean values and the standard e r r o r o f the mean ( v e r t i c a l bars) o r as percentage o f c o n t r o l values. RESULTS D i u r e t i c A c t i v i t y i n Mice - As shown i n Figure 2, the o r a l admin­ i s t r a t i o n o f t i e n i l i c a c i d (20, 50, 100, 200 mg/kg p.o.) gave a s i g n i f i c a n t i n c r e a s e i n u r i n e volume and e l e c t r o l y t e e x c r e t i o n . However, i n comparison with furosemide o r e t h a c r y n i c a c i d , t i e n i l i c a c i d i s about 1/5 to 1/10 as potent. Nevertheless, contrary to other d i u r e t i c and n a t r i u r e t i c agents, t i e n i l i c a c i d i n c r e a s e s urate e x c r e t i o n . The u r i c o s u r i c a c t i v i t y o f t i e n i l i c a c i d i n mice i s doser e l a t e d , as shown i n F i g u r e 3, i n comparison with that o f f u r o s e ­ mide and e t h a c r y n i c a c i d i n e f f e c t i v e d i u r e t i c doses. I n the same experiment, benziodarone, which possesses s i g n i f i c a n t u r i c o s u r i c

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

BESsiN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

.01<*< . 0 5 •001<»t< .01

|*j AN Ρ3824 2 0 . 5 0 , 1 0 0 , 2 0 0 mg/kg RO. S T U D E N T ' S t TEST

I^JEthacrynic a c i d , 2 0 mg/kg R O . j°J F u r o s e m i d e , 2 0 m g / k g P.O. [^J B e n z i o d a r o n e , 1 0 0 m g / k g P.O.

H 3

1

J

Diuresis

Figure 2.

CI

ANΡ 3624 (tienilic acid-ticrynafen)—diuretic and uricosuric activity in the mouse • P < 05 • Ρ < 05 0,0,#N.S.

_ J_B e η z ip_d_a rone

J.

J. 20

50 D O S E

100

200

m g / k g P.O.

Figure 3. ANP 3624 (tienilic acid-ticrynafen)— uricosuric activity in the mouse

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

62

DIURETIC AGENTS

p r o p e r t i e s i n humans (46,67), has increased the urate e x c r e t i o n i n mice without g r e a t l y a l t e r i n g r e n a l f u n c t i o n . However, i n the b e n z i o d a r o n e - t i e n i l i c a c i d i n t e r r e l a t i o n s h i p s t u d i e s , i t was demonstrated that benziodarone i n h i b i t s the d i u r e t i c e f f e c t of t i e n i l i c a c i d , but t h e i r u r i c o s u r i c e f f e c t s were s y n e r g i s t i c (Figure 4 ) . Conversely, the benziodarone-furosemide combination does not modify these parameters. As seen i n Table I , t i e n i l i c a c i d i s the most a c t i v e member of t h i s s e r i e s , and only three compounds possess both d i u r e t i c and u r i c o s u r i c a c t i v i t i e s , i . e . , ANP 3624 ( t i e n i l i c a c i d ) , ANP 4316 (the 3 - t h i e n y l analogue of t i e n i l i c a c i d ) and ANP 3860 (the 2-benzofuranyl analogue of t i e n i l i c a c i d ) . T h i s l a t t e r compound, which produced an i n c r e a s e i n urate e l i m i n a t i o n along with a weak n a t r i u r e t i c e f f e c t , possesses some of the s t r u c t u r a l f e a t u r e s of the 2,3-dichlorophenoxyacetic a c i d s and some of those of benziodarone, which i s obvious that t i e n i l i c a c i transport. T h i s a c t i v i t y of the drug, i n i t i a l l y observed i n mice, has been confirmed i n r a t s and dogs. O r a l D i u r e t i c A c t i v i t y of T i e n i l i c A c i d i n the Rat - As seen i n Figure 5 and according to many authors, e t h a c r y n i c a c i d was i n e f f e c t i v e at a dose as high as 300 mg/kg (Γ = .29 NS); tienilic a c i d was weakly d i u r e t i c i n doses up to 400 mg/kg Jj = .96 (P < .0517 and furosemide e x h i b i t e d marked d i u r e t i c a c t i v i t y a t a dose of 50 mg/kg. U r i c o s u r i c A c t i v i t y of T i e n i l i c A c i d i n the Rat as Measured by the Phenol-Red Test - In view of the d i f f i c u l t i e s i n a d m i n i s t e r i n g u r i c a c i d to s m a l l animals, the i n d i r e c t method of Scarborough and McKinney (41) f o r s c r e e n i n g u r i c o s u r i c agents i n r a t s was used. T h i s i n d i r e c t method does not measure the e x c r e t i o n of u r i c a c i d , but r a t h e r the phenol-red r e t e n t i o n induced by a l l u r i c o s u r i c substances. In t h i s t e s t , as i l l u s t r a t e d i n Figure 6, t i e n i l i c a c i d and benziodarone e x h i b i t e d s i m i l a r i n h i b i t i o n of phenol-red e l i m i ­ n a t i o n , while furosemide and e t h a c r y n i c a c i d were i n e f f e c t i v e . I t should be noted that i n the same experiment probenecid and s u l f i n ­ pyrazone reduced the phenol-red e l i m i n a t i o n and t h a t a c e t y l s a l i c y l i c a c i d s t r o n g l y i n h i b i t e d the a c t i v i t y of t i e n i l i c a c i d and benziodarone (unpublished data). D i u r e t i c A c t i v i t y i n A n e s t h e t i z e d Beagle Dogs - D i u r e t i c and N a t r i u r e t i c A c t i v i t y - The acute e f f e c t of t i e n i l i c a c i d on u r i n e and e l e c t r o l y t e e x c r e t i o n i n a n e s t h e t i z e d beagle dogs i s i l l u s ­ t r a t e d i n F i g u r e s 7-11. Intravenous a d m i n i s t r a t i o n o f 5, 10, 20 mg/kg of t i e n i l i c a c i d caused a s t a t i s t i c a l l y s i g n i f i c a n t doser e l a t e d e x c r e t i o n of u r i n e (Figure 7) with an i n c r e a s e of r e n a l sodium (Figure 8 ) , potassium (Figure 9) and c h l o r i d e e x c r e t i o n (Figure 10). At every dose, the sum of Na and Κ excretion was

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

BESsiN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

Figure 4. Antagonism benziodarone-AN Ρ 8624 (diuresis) with synergistic increase of the uricosuric effect (mice Ν =6)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

Table I

DIURETIC ANP

ACTIVITY

(MICE)

M κ·

Na*

H ET.

urates

3624 1

3649

1.2

1.8

1.1

1.7

4357

8

.7

.6

1.2

.9

1.8*

5.4*

1.5'

3.5

1.1

43Q4

1.1

1.4

8

1.7

1.1

4316

2.7*

2.2-

1.9

1.2

1.6*

4381*

1.6*

2

1

1.8*

.9

1.2

1.2

9

1.3

1.1

4294

1.3

i.a

1.2

1.1

1.3

3860*

1.3

1.5

• 1.7

.9

1.6*

2.4*

2.8

1.5

1.9*

13

3*

4.3*

1.5

2B*

1.4

3598*

4303

A* B

#

UL

0^ ETACRYNIC ACID FUROSEMIDE

•

e

. , « , * , Ρ < 0 . 0 5 STUDENT'S t T E S T Α,Β

20mg/kg

AN Ρ 3624...

PO

100 mg/kg

PO

European Journal of Medicinal Chemistry

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

5.

BESSIN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

4

1

10

20

1

' 50

100

» 200

â 300

» 400 mg/kg

Figure 5. ANP 3624 (tienilic acid-ticrynafen)—diuretic activity in the normal rat

Τ IM E( M I N U T E S )

Figure 6.

AN Ρ 3624 (tienilic acid-ticrynafen)—retention of phenol red in the rat (N = 5)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

65

DIURETIC AGENTS

66

m E q / 15mn

!"^'."Z7J

AU

P < 0.05/To (TUKEY'S

A N P ^ 3 6 2 4 | 20mg/kg

ff

I.V. (N = 6 )

V

χ

1

if

TEST)

Μ ι V

N!

1ANP I 3 6 2 4

χ

10mg/kg W . ( N = 4 )

/.(N=5)

- τ ι V

Figure 8.

I

1

-A

i

I

ANP 3624 1

^

5mg/kg ^

^

- T —

r

I CONTROLS

•>—I—

I.V.(N=5) 2h * *

r

^

Γ I ^

-Λΰ»

1

Î

PLACEBO

I

T

'

J

( N = 5) N ^ 3h

•

ANP 3624 (tienilic acid-ticrynafen)—diuretic activity in the anesthetized beagle dog (Na*)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

5.

BESsrN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

1

1

1 T

Figure 9.

1

1

1

V

0

1

1

1——I

1

"

67

ι

2H.

I 3H.

ANP 3624 (tienilic acid-ticrynafen)—diuretic activity in the anesthetized beagle dog (K*)

Figure 10.

ANP 3624 (tienilic acid-ticrynafen)—diuretic activ­ ity in the anesthetized beagle dog (Cl~)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

68

about equal to t h a t of c h l o r i d e e x c r e t i o n and the u r i n a r y pH remained unchanged. In these experiments, the t o t a l duration of urine and e l e c t r o l y t e e x c r e t i o n was approximately 3 hours. A f t e r intravenous a d m i n i s t r a t i o n , the maximal d i u r e t i c and n a t r i u r e t i c response occurred w i t h i n 30 minutes a f t e r drug i n j e c t i o n . In a l l of the experiments the r i s e o f Na e x c r e t i o n was accompanied by a r i s e i n K e x c r e t i o n , but the Na r a t i o was g e n e r a l l y increased as with each of the d i u r e t i c substances (Figure 11). +

H

U r i c o s u r i c A c t i v i t y - The e f f e c t s of t i e n i l i c a c i d on the urate e x c r e t i o n i n anesthetized beagle dogs are seen i n Figures 12-14. Intravenous a d m i n i s t r a t i o n o f 10 and 20 mg/kg of t i e n i l i c a c i d caused a s t a t i s t i c a l l y s i g n i f i c a n t urate e x c r e t i o n . The time o f maximum increase i n u r i n e output i s about the same as that f o r Na e x c r e t i o n and the greates minutes a f t e r treatment l a s t e d f o r 3 hours (Tukey's t e s t ) . In t h i s experiment, 5 mg/kg of t i e n i l i c a c i d d i d not induce u r i c o s u r i a . During the same p e r i o d , no s t a t i s t i c a l l y s i g n i f i c a n t changes i n c r e a t i n i n e clearance were noted (Figures 13, 14). Clearance Studies - As shown i n Figures 13-15, t i e n i l i c a c i d a t i . v . doses of 5, 10 and 20 mg/kg a f f e c t e d r e n a l clearances, p a r t i c u l a r l y osmolar clearance and f r e e water reabsorption when exogenous c r e a t i n i n e e x c r e t i o n showed no s t a t i s t i c a l l y s i g n i f i c a n t change. In the hydropenic s t a t e , a s l i g h t r i s e of urine volume was accompanied by marked increases i n t o t a l solute clearance. T h i s e f f e c t , which i s shown i n Figure 15 (20 mg/kg i . v . of t i e n i l i c a c i d ) , caused an increase i n f r e e water reabsorption. But, i n t e r ­ e s t i n g l y , when these data were c o r r e c t e d f o r osmolar clearance and glomerular f i l t r a t i o n r a t e , as p r e v i o u s l y suggested (45, 47-52), t i e n i l i c a c i d proved to decrease the negative f r e e water clearance (Figure 16), i . e . , i t decreases f r e e water reabsorption. In these experiments, a f t e r 20 mg/kg i . v . of t i e n i l i c a c i d (N = 6), osm increased from 0.60 +0.11 to 1.79 +0.26 ml pgr minute a f t e r 30 minutes when there was a progressive r i s e inJT H^O from 0.40 + 0.07 to 0.80 +0.11. Using data f a c t o r e d by osm and glomerular f i l t r a t i o n rate (Figure 16), Τ HJD decreased from 3·527 + 1.028 to 1.410 +0.366 a f t e r 90 minutes. In the same experiment, 10 mg/kg of t i e n i l i c a c i d provoked a s i m i l a r decrease i n c o r r e c t e d tubular water reabsorption, i . e . , dropping from 3-103 + 1.258 to 0.918 + 0.268 a f t e r 60 minutes. These disturbances i n r e n a l concentrating mechanism, which are summarized i n Figure 16 and s t a t i s t i c a l l y analyzed by Tukey's t e s t , were s t i l l s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l values 3 hours a f t e r the d i u r e t i c was given; thus, the a d m i n i s t r a t i o n of 5 mg/kg of t i e n i l i c a c i d l e d to a s i g n i f i c a n t change i n the f r e e water r e a b s o r p t i o n .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

BESSiN ET AL.

Figure 11.

TieniUc Acid in Mice, Rats, and Beagles

ANP 3624 (tienilic acid-ticrynafen)—diuretic tized beagle dog

Figure 12.

activity in the anesthe-

ANP 3624 (tienilic acid-ticrynafen)—uricosuric anesthetized beagle dog

activity in the

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

70

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In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Tienilic Acid in Mice, Rats, and Beagles

71

Figure 16. ANP 3624 (tienilic acid-ticrynafen)—diuretic activity in the anesthetized beagle dog

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

72

DIURETIC AGENTS

During the same p e r i o d , minor v a r i a t i o n s i n PAH, c r e a t i n i n e and urea clearances were induced by 10 and 20 mg/kg o f t i e n i l i c a c i d as demonstrated i n F i g u r e s 13 and 14. Notably, the clearance of PAH i n c r e a s e d from 71.38 + 11.22 to 100.04 + 29-99 and from 66.4 + 13.03 to 121.27 + 39.16 a t 5 mg/kg and 20 mg/kg o f t i e n i l i c a c i d , r e s p e c t i v e l y . Under the same experimental c o n d i t i o n s , the glomerular f i l t r a t i o n r a t e s l i g h t l y increased, i . e . , the f i l ­ t r a t i o n f r a c t i o n had a value between 0.35 and 0.50, o r remained unchanged. The time r e q u i r e d f o r the maximum change i n PAH clearance was u s u a l l y about 15-30 minutes a f t e r a d m i n i s t r a t i o n o f t i e n i l i c acid. A n t i h y p e r t e n s i v e A c t i v i t y o f T i e n i l i c A c i d i n the SH Rat - The combined d i u r e t i c , n a t r i u r e t i c and u r i c o s u r i c p r o p e r t i e s o f t i e n i l i c a c i d l e d to a stud f i t p o t e n t i a l antihypertensiv a c t i v i t y i n SH r a t s usin i n which the c l a s s i c c u f Acute A n t i h y p e r t e n s i v e A c t i v i t y (Figure 17) - The s y s t o l i c blood pressure o f SH r a t s was s i g n i f i c a n t l y (Tukey's t e s t ) decreased a f t e r 2, 5 and 24 hours f o l l o w i n g a d m i n i s t r a t i o n o f 200 and 400 mg/kg o f t i e n i l i c a c i d by the o r a l r o u t e . The placebo d i d not produce any change i n blood pressure, and t i e n i l i c a c i d a t 100 mg/kg s i g n i f i c a n t l y decreased the blood pressure only a f t e r 2 and 5 hours f o l l o w i n g a d m i n i s t r a t i o n o f the drug. Subacute A n t i h y p e r t e n s i v e A c t i v i t y - Figure 18 shows that d a i l y a d m i n i s t r a t i o n o f 200 mg/kg o f t i e n i l i c a c i d f o r 5 days produced a s u s t a i n e d a n t i h y p e r t e n s i v e response i n SH r a t s a t each time i n t e r v a l during the experiment. Three days a f t e r withdrawal o f the drug, the blood pressure returned to c o n t r o l v a l u e s . Chronic A n t i h y p e r t e n s i v e A c t i v i t y (Figure 19) - T i e n i l i c a c i d was administered o r a l l y to SH r a t s i n d a i l y doses o f 100 and 200 mg/kg (mixed with food) f o r 6 months. F o r 100 mg/kg, Ν = 27; 200 mg/kg, Ν = 29; c o n t r o l normal r a t s , (N.R.) Ν = 15 and c o n t r o l SH r a t s (SHR) Ν = 25. The s y s t o l i c blood pressure was measured a t the beginning and then a f t e r 1.5, 3.5 and 6 months. As i l l u s t r a t e d i n F i g u r e 19, t i e n i l i c a c i d (200 mg/kg) decreased the s y s t o l i c blood pressure (P<0.05 - Tukey's t e s t ) i n SH r a t s a f t e r 3-5 and 6 months. Neither c o n t r o l nor t i e n i l i c a c i d a t 100 mg/kg s i g n i f ­ i c a n t l y a f f e c t e d the blood pressure measured i n d i r e c t l y i n SH rats. I n t e r e s t i n g l y , a f t e r 20 months, the s y s t o l i c blood pressure was not s i g n i f i c a n t l y a l t e r e d by e i t h e r 100 o r 200 mg/kg o f t i e n i l i c acid. However, the m o r t a l i t y o f the SH r a t s was s t a t i s ­ t i c a l l y l e s s i n the a n i m a l s _ r e c e i v i n g 200 mg/kg of t i e n i l i c a c i d , τ|γ = 408; 0.01 < α < 0.05 ^Wilcoxon's non-parametric procedure (53)7· The r e s p e c t i v e numbers o f s u r v i v i n g animals were 12/15 (normotensive Wistar r a t s ) , 4/21 ( c o n t r o l SH r a t s ) , 16/27

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

5.

BESSiN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

c m Hg 20 J

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2

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Figure 17. ANP 3624 (tienilic acid-ticrynafen)—acute antihypertensive activity in the SH rat (N = 6)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

73

DIURETIC AGENTS

74

Figure 19.

ANP 3624 (tienilic acid-ticrynafen)—antihypertensive activity. Chronic study in the SH rat (S).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

5.

BEssiN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

( t i e n i l i c a c i d at 200 mg/kg) and 5/26 mg/kg).

75

( t i e n i l i c a c i d a t 100

Blood T r i glyceride-Lowering E f f e c t i n the Obese Rats ( F a t t y S t r a i n ) - In previous experiments, Maass (£4) found t h a t t i e n i l i c a c i d decreases the blood l e v e l s of t r i g l y c e r i d e s i n normal r a t s . A s i m i l a r a c t i v i t y has been found again i n obese r a t s ( F a t t y S t r a i n ) , but not i n normal r a t s (Figure 20). However, t o t a l l i p i d s , blood c h o l e s t e r o l and glucose l e v e l s remained unchanged i n both animal models. DISCUSSION T h i s paper d e s c r i b e s the d i u r e t i c , n a t r i u r e t i c and u r i c o s u r i c studies c a r r i e d out over a p e r i o d o f years (28) i n a v a r i e t y o f animals, i n c l u d i n g mice and c l i n i c a l r e p o r t s hav (54-63) o f Masbernard ( £ 2 ) who f i r s t reported the human pharmac o l o g i c a l studies on t h i s drug. As shown i n mice, r a t s and a n e s t h e t i z e d beagle dogs, t i e n i l i c a c i d (ANP 3624), i n c o n t r a s t to furosemide, e t h a c r y n i c a c i d , the indanone and bumetanide, i s a m i l d s a l u r e t i c - d i u r e t i c agent a t r e l a t i v e l y high doses. But, i n a l l the s p e c i e s studied i n these experiments, t i e n i l i c a c i d e x h i b i t e d the potent u r i c o s u r i c act i v i t y required to decrease hyperuricemia, which i s a c a r d i o vascular r i s k factor. In mice, the d i u r e t i c , u r i c o s u r i c and n a t r i u r e t i c a c t i v i t i e s of t i e n i l i c a c i d were found to be dose-dependent (Figure 3); i n normal r a t s , t i e n i l i c a c i d , i n c o n t r a d i s t i n c t i o n to e t h a c r y n i c a c i d , provoked a d o s e - r e l a t e d d i u r e t i c and n a t r i u r e t i c response. In the same animal, t h i s d i u r e t i c agent decreased phenol-red e l i m i n a t i o n , which has been regarded as an i n d i r e c t measurement o f a competitive antagonist o f urate e x c r e t i o n i n dogs, o c c u r r i n g mainly i n the proximal tubule ((54,65,66). T h i s p o t e n t i a l u r i c o s u r i c a c t i v i t y as measured i n the r a t by antagonism o f phenol-red e l i m i n a t i o n has been demonstrated by Lemieux (62) i n a r e c e n t r e p o r t which r e v e a l s that t i e n i l i c a c i d provoked a u r i c o s u r i c e f f e c t i n man, mouse, r a t and dog ( a l l s p e c i e s where urate reabs o r p t i o n predominates). Indeed, i n a n e s t h e t i z e d beagle dogs, under hydropenic c o n d i t i o n s , t i e n i l i c a c i d e x h i b i t s both d i u r e t i c and u r i c o s u r i c p r o p e r t i e s over a wide dose range. The d i u r e t i c a c t i v i t y was d i s t i n g u i s h e d by a marked i n c r e a s e i n water and N a e l i m i n a t i o n with s i g n i f i c a n t K and C l " excret i o n . But, i t was o f i n t e r e s t to note t h a t clearance s t u d i e s showed a frank r i s e i n both urate and osmolar c l e a r a n c e s . T h i s renders t i e n i l i c a c i d unique among the known d i u r e t i c agents, with the exception of another a r y l o x y a c e t i c a c i d d e r i v a t i v e , i . e . , the indanone which a l s o e x h i b i t e d potent d i u r e t i c and u r i c o s u r i c a c t i v i t i e s i n chimpanzees and human s u b j e c t s (32,^3,^4,35). As demonstrated i n previous r e p o r t s by other authors (54,60), +

+

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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ANP 3624 (tienilic acid-ticrynafen)—200 mg/kg daily po (5 days)—study on the blood triglycerides lowering effect in the obese rat (fatty strain)

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Tienilic Acid in Mice, Rats, and Beagles

77

t i e n i l i c a c i d a f f e c t s reabsorption and s e c r e t i o n o f urate i n dogs mainly i n the proximal tubule. Other s t u d i e s concerning the s i t e of a c t i o n o f s e v e r a l n o n - d i u r e t i c u r i c o s u r i c agents suggested the same c o n c l u s i o n s (65,66,67,68). I n h i b i t i o n o f phenol-red excre­ t i o n i n r a t s by u r i c o s u r i c agents and by d i u r e t i c - u r i c o s u r i c substances i n d i c a t e s t h a t they s p e c i f i c a l l y depress urate reabs o r p t i o n a t the proximal s i t e (64,65,66). On the other hand, as summarized i n Figure 15, t i e n i l i c a c i d e x h i b i t e d a g r e a t e r i n c r e a s e i n osmolar clgarance than i n water excretion. Consequently, the c a l c u l a t e d Τ H O was s i g n i f i c a n t l y r a i s e d , as reported by S t o t e et a l (5j4) and to a l e s s e r degrge by Maass et a l (59). In c o n j u n c t i o n with the previous data on H^O, t i e n i l i c a c i d produced a l t e r a t i o n s i n the c o r t i c a l d i l u t i n g segment of the d i s t a l nephron, u n l i k e the other loop d i u r e t i c s . However, when the r e a b s o r p t i o n o f s o l u t e free-water (Τ Η,,Ο) was f a c t o r e d by osmolar clearanc rate ( c r e a t ) as p r e v i o u s l r e a b s o r p t i o n was s u r p r i s i n g l y decreased by the a d m i n i s t r a t i o n o f t i e n i l i c a c i d ^ ( F i g u r e s 15 and 16). A s i m i l a r r e v e r s a l was ob­ tained when Τ H^O was c o r r e c t e d f o r osmolar clearance alone, a t t e n u a t i n g the p o s s i b l e prominent p a r t played by the c r e a t i n i n e c l e a r a n c e r a t e on the genesis of t h i s phenomenon. Jn the f i r s t absolute c a l c u l a t i o n o f Τ H 0 as i t obtained from osm, the a c t i o n of t i e n i l i c a c i d was q u a l i t a t i v e l y s i m i l a r to t h a t of c h l o r o t h i a z i d e . Using the c o r r e c t e d Τ H 0, the t i e n i l i c a c i d r e s u l t s were approximately s i m i l a r to those o f e t h a c r y n i c a c i d and furosemide, while they were opposite to those of the t h i a z i d e s . C o n s i d e r i n g these r e s u l t s i n the l i g h t of the p r o p e r t i e s of t i e n i l i c a c i d i n a hydrated s t a t e , these c o r r e c t e d c a l c u l a t i o n s o f Τ H 0 do not exclude the p o s s i b i l i t y t h a t t i e n i l i c a c i d could a l t e r an a d d i t i o n a l s i t e i n the r e n a l tubule. In an attempt to support t h i s assumption* i t i s i n t e r e s t i n g to note t h a t the c a l c u l a t i o n of both c o r r e c t e d H 0 and Τ H 0 from data r e ­ ported i n papers from s e v e r a l i n v e s t i g a t o r s , no change i n the normal increase o f h y d r o c h l o r o t h i a z i d e a c t i v i t y on s o l u t e - f r e e water r e a b s o r p t i o n (69) was seen. On the other hand, the c o r r e c t e d c a l c u l a t i o n s reversed the r a t h e r m i l d e f f e c t of t i e n i l i c a c i d on Τ H Q s i m i l a r to t h a t seen i n the present study (£4) and r e s t o r e d the H 0 to the expected values f o r furosemide (70,71) and c h l o r merodnne (72) which, d i s p l a y e d p a r a d o x i c a l l y , a l t e r e d absglute 2

2

2

2

2

2

2

gpO values. More e x a c t l y , the c o r r e c t e d c a l c u l a t i o n s of H 0 and Τ HpO have normalized the p a r a d o x i c a l e f f e c t s observed f o r f u r o ­ semide or chlormerodrine, while the same c o r r e c t i o n s reversed the s i g n i f i c a n c e of the e f f e c t s of t i e n i l i c a c i d i n regard to s o l u t e f r e e water r e a b s o r p t i o n . F o r example, i n the experiment gf Stote et a l (54), t i e n i l i c a c i d very moderately i n c r e a s e d the Τ H 0 before the c o r r e c t i o n , and s i g n i f i g a n t l y decreased the Τ H^O value u s i n g the same c a l c u l a t i o n , while H 0 i n water d i u r e s i s was not modified when an analogous c a l c u l a t i o n was made with respect to u r i n a r y flow and i n u l i n c l e a r a n c e . In the same way, i n the r e p o r t 2

2

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Journal de Pharmacologie Clinique Figure 21. Tienilic acid (250-750 mg daily)—antihypertensive and uricosuric effect in essential hypertensive patients (N = 12) (64)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

BEssiN ET AL.

Tienilic Acid in Mice, Rats, and Beagles

Figure 22. Tienilic acid—effect on uric acid in man—serum values and urinary excretion (54)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

80

of Suki et a l (70), using furosemide at 2 mg/kg i . v . paradoxically increased the calculated HO in dogs but markedly decreased the experimental value. However, i n the report of Azer et a l (71), which describes the proximal tubular rejection of sodium gy furosemide, chlormerodrine, 8 mg/kg, did not change the Τ H 0 because of paradoxical data generated by his three experiments; but after correction, chlormerodrine depressed significantly solute-free water reabsorption in comparison to the control value. In conclusion, i t i s suggested that these data are consistent with the idea that under some conditions of urinary excretion, t i e n i l i c acid possesses an additional site of action. On the other hand, i t is of interest to observe that there is a good parallelism between the animal data and the c l i n i c a l studies, as shown in Figures 21 and 22, which confirm the diuretic, uricosuric and antihypertensive propertie f t i e n i l i acid i It should be noted that th observed in small rodent dogs Finally, the combined diuretic, uricosuric, antihypertensive and triglyceride-lowering properties of t i e n i l i c acid observed in rats suggest that the control of these factors as risks in cardiac diseases may explain the increase of survival time of SH rats after chronic administration of t i e n i l i c acid. Literature Cited 1. Schultz, E . M . , Cragoe, E . J., Jr., Bicking, J. B . , Bolhofer, W. A. and Sprague, J. M . , J. Med. Pharm. Chem. (1962), 5, 660. 2. Baer, J. E., Michaelson, J. K . , McKinstry, D. N. and Beyer, K. H . , Proc. Soc. Exp. B i o l . Med. (1964), 115, 87-90. 3. Kleinfelder, H . , Ger. Med. (1963), 8, 459. 4. F e i t , P. W., J. Med. Chem. (1971), 14, 432. 5. Feit, P. W. and Tvaermose Nielsen, O. B . , J. Med. Chem. (1972), 15, 79. 6. Wilkins, R. W., Ann. Int. Med. (1959), 50, 1. 7. Goldner, M. G. H . , Zarowitz, H . , Akgun, S., N. Engl. J. Med. (1960), 262, 403. 8. Shapiro, A. P . , Benedek, T. G . , Small, J. L., N. Engl. J. Med. (1960), 265, 1028. 9. Lyon, A. F., DeGraff, A. C., Am. Heart J. (1964), 68, 710. 10. Hartmann, F . and Heimsoth, V . , "Antihypertensive Therapy", 436-447. Proceedings edited by F . Gross, Springer-Verlag, 1966. 11. DeMartini, F . E., A r t h r i t i s and Rheum. (1965), 8, 823-829. 12. Carmon, P. J., Heinemann, H. O., Stason, W. B . , Laragh, J. Η., Circulation (1965), 31, 5. 13. Stason, W. B . , Carmon, P. J., Heinemann, H. O. and Laragh, J. H . , Circulation (1966), 34, 190-200. 14. Humphreys, M. B . , Br. Med. J. (1966), 1, 1024-1025. 15. McKenzie, F . C., Fairley, K. F., Baird, C. W., Med. J. Aus. (1966), 879-886. 16. Hutcheon, D. E., Mehta, D . , Romano, Α . , Arch. Int. Med. Exp. (1965), 115, 542-546. 17. Bryant, J. Μ., Fan Yu, T . , Berger, L., Schwartz, Ν . , c

2

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Torosdag, S., Fletcher, L., Fertig, H . , Schwartz, S., Quan, F . B. F., Am. J. Med. (1962), 33, 408. 18. Kim, Κ. E., Onesti, G . , Mayer, J. H . , Schwartz, C., Am. J. Cardiol. (1971), 27, 407. 19. Olesen, Κ. Η., Sigurd, B . , Steiness, E., Leth, Α . , Acta Med. Scand. (1973), 193, 119-131. 20. Steele, T. H . , Rheumatic Diseases (1977), 3, 37-50. 21. Bourke, E., Asbury, M. J. Α . , O'Sullivan, S. and Gatenby, P. Β. B . , Eur. J. Pharmacol. (1973), 23, 283-289. 22. Davies, D. L., Lant, A. F., Millard, Ν. R . , Smith, A. J., Ward, J. W. and Wilson, G. Μ., C l i n . Pharmacol. Ther. (1973), 15, 141-155. 23. Hutcheon, D. Ε., Pocelinke, R. and Duchin, K. L., "New Antihypertensive Drugs", 323-336, A. Scriabine and C. S. Sweet. Spectrum Publications, Inc. New York 1976 24. Wiebelhaus, V. D . G. and Larsen, T. J., Fed 25. Baer, J. E., Jones, C. B . , Spitzer, S. A. and Russo, H. F., J. Pharmacol. Exp. Ther. (1967), 157, 472-485. 26. French Patent 2068403 (1973). 27. Godfroid, J. J. and T h u i l l i e r , J. Ε., U.S. Patent 3,758,506 (1973). 28. T h u i l l i e r , G. Laforest, J., Cariou, B. Bessin, P . , Bonnet, J. and T h u i l l i e r , J., Eur. J. Med. Chem. Chim. Ther. (1974), 9, 625-633. 29. Masbernard, A. and G u i d i c e l l i , C., Lyon Med. (1974), 232, 165-174. 30. Stote, R. M . , C h e r r i l l , D. Α . , Erb, Β. B. and Alexander, F., C l i n . Res. (1974), 22, 721A. 31. Stote, R. M . , C h e r r i l l , D. Α . , Maass, A. R., Erb, Β. B . , Familiar, R. G. and Alexander, F., Abs. 6th Int. Cong. Neph., Florence, Italy, (1975) 827. 32. Cragoe, E. J., Jr., Schultz, Ε. Μ., Schneeberg, J. D . , Stokker, G. E., Woltersdorf, O. W., F a n e l l i , G. M. and Watson, L . S., J. Med. Chem. (1974), 18, 225-228. 33. Woltersdorf, O. W., Schneeberg, J. D., Cragoe, E. J., Jr., Schultz, Ε. M . , Stokker, G. E., Watson, L . S. and F a n e l l i , G. M . , Abs. 169th Am. Chem. Soc. Nat. Mtg., Philadelphia, P a . , (1975) No. 49. 34. Woltersdorf, O. W., Cragoe, E . J., Jr., Watson, L . S. and F a n e l l i , G. M . , Abs. 169th Am. Chem. Soc. Nat. Mtg., Philadelphia, P a . , (1975) No. 48. 35. Watson, L . S., F a n e l l i , G. Μ., Russo, H. F., Sweet, C. S., Ludden, C. T., Scriabine, Α . , "New Antihypertensive Drugs", 307-321, A. Scriabine and C. S. Sweet, Spectrum Publications, Inc., New York, 1976. 36. Bessin, P . , Tetu, O. and Selim, Μ., Chim. Ther. (1969), 4 (3), 220. 37. Schales, O. and Schales, S., J. B i o l . Chem. (1941), 140, 879. 38. Kageyama, N . , C l i n . Chim. Acta. (1971), 31 (2), 421.

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39. Caraway, W. T., Am. J. C l i n . Pathol. (1955), 25, 840. 40. Kreppel, E., Med. Exp. (1959), 1, 285. 41. Scarborough, H. C. and McKinney, G. R . , J. Med. Pharm. Chem. (1962), 5, 175. 42. Hamburger, J., Ryckewaert, Α . , Duysent, M. and Argant, N . , Ann. B i o l . C l i n . (Paris) (1948), 6, 358. 43. Popper, H., Mandel, E. and Mayer, H., Biochem. Z. (1937), 291, 354. 44. Fawcett, J. K. and Scott, J. E., J. C l i n . Pathol. (1960), 13, 156. 45. Carriere, S. and Dandavino, R., C l i n . Pharmacol. and Ther. (1976), 20 (4), 424-438. 46. Nivet, M . , Marcovici, J. and Laruelle, F., B u l l . Mem. Soc. Med. Hop. Paris (1965), 116, 1187. 47. Goldberg, M . , McCurdy, D. Κ., Foltz, E . L . and Bluemle, L . W., J. C l i n . Invest. (1964), 43, 201-216. 48. Suki, W., Rector, F C., J r and Seldin D W. J. C l i n Invest. (1965), 44, 1458-1469 49. Stein, J. H., Wilson C l i n . Med. (1968), 71, 654-665. 50. Puschett, J. B. and Goldberg, M . , J. Lab. C l i n . Med. (1968), 71, 666-677. 51. Martinez-Maldonado, M . , Suki, W. Ν. and Schenker, S., Am. J. Physiol. (1969), 216, 1376-1391. 52. Bourke, E., Asbury, M. J. Α . , O'Sullivan, S. and Gatenby, P. Β. B . , Eur. J. Pharmacol. (1973), 23, 283-289. 53. Wilcoxon, F . and Wilcox, R. Α . , "Some Rapid Approximate S t a t i s t i c a l Procedures", American Cyanamid Company, Pearl River, New York, 1964. 54. Stote, M . , Maass, A. R., C h e r r i l l , D. Α . , Beg, M. M. A. and Alexander, F., J. Pharmacol. C l i n . (1976), Special Issue, 19-27. 55. Masbernard, Α . , G i u d i c e l l i , C. P. and Kamaludin, T., J. Pharmacol. C l i n . (1976), Special Issue, 13-18. 56. Reese, O. G. and Steele, T. H., Am. J. Med. (1976), 60, 973-979. 57. Jain, Α. Κ., Ryan, J. R. and McMahon, F . G . , C l i n . Res. (1876), 24,1. 58. Steele, T. H., Prasad, D. R. and Reese, O. G . , C l i n . Pharmacol. Ther. (1976), 19, 116. 59. Maass, A. R., Erickson, R., Snow, I., Brennan, F., Weedon, R. and Hutchings, G . , Physiologist (1976), 19, 280. 60. Stote, R . , Goldberg, M. and Agus, Z. S., C l i n . Res. (1976), 24, 405A. 61. G i l l i e s , Α . , Morgan, G. and Morgan, T., Aust. N. Z. J. Med. (1977), 7, 443-444. 62. Nemati, M. Kyle, M. C. and Freis, E . D . , J. Am. Med. Assoc. (1977), 237, 652-657. 63. Lemieux, G . , Kiss, Α . , Vinay, P. and Gougoux, Α . , Kidney Int. (1977), 12, 104-114. 64. Springinsfeld, Μ., Durel, J., R i e f f e l , R . , and Jahn, H., J. Pharmacol. Clin. (1976), Special Issue, 35-45. 65. Marshall, Ε. K . , Am. J. Physiol. (1931), 99, 77.

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66. Zins, G. R. and Weiner, I . Μ., Am. J. Physiol. (1968), 215, 411-422. 67. Weiner, I . M. and F a n e l l i , G. M . , "Recent Advances in Physiology and Pharmacology", 53-68, Wessin, L . G. and F a n e l l i , G. M . , Eds., University Park Press, 1974. 68. Lemieux, G., Gougoux, Α . , Vinay, P. and Michaud, G., Am. J. Physiol. (1973), 224, 1431-1439. 69. Lemieux, G., Vinay, P . , Gougoux, A. and Michaud, G., Am. J. Physiol. (1973), 224, 1440-1449. 70. Early, L . E., Kahn, M. and Orloff, J., J. C l i n . Invest. (1961), 40, 857-866. 71. Suki, W. Ν., Rector, F . C. and Seldin, D. W., J. C l i n . Invest. 1965), 44, 1458-1469. 72. Azer, M. and Kirkendal, W. M . , J. Pharmacol. Exp. Ther. (1973), 185, 235-244. 73. Puschett, J. B. an 71, 666-677. 74. Maass, A. R., private communication. RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

6 Ticrynafen: An Antihypertensive, Diuretic, Uricosuric Agent A. R. MAASS, I. B. SNOW, and R. ERICKSON Smith Kline and French Laboratories, 1500 Spring Garden St., Philadelphia, PA 19101 R. M . STOTE Presbyterian-University of Pennsylvania Medical Center, Philadelphia, PA 19104

It is apparent tha publication that i t is i c a l structures which are modifications of existing diuretics in anticipation of obtaining a compound with similar diuretic activity. Examples of diuretics with markedly different chemical structures such as: mersalyl (1), ethacrynic acid (2), furosemide (3) and bumetanide (4), a l l of which act at the same site in the renal tubule have been known for some time. However, we also now know of two dichlorophenoxyacetic acid diuretics which act at different sites within the kidney tubule. Ticrynafen is [2,3-dichloro-4-(2-thienylcarbonyl)phenoxy]acetic acid. It was registered with the World Health Organization as t i e n i l i c acid; the USAN approved name, however, i s ticrynafen. On the basis of i t s dichlorophenoxyacetic acid structure, i t appears to be related to ethacrynic acid, [2,3-dichloro-4-(2-methylenebutyryl)phenoxy]acetic acid, but is chemically and pharmacologically quite different. For example, ethacrynic acid reacts avidly in vitro with the sulfhydryl amino acid, cysteine, while ticrynafen is quite inert. Since Burg (5) and others (6) have shown that i t is the ethacrynic acid-cysteine adduct which is the active diuretic (at least in v i t r o ) , we were led to the conclusion that the two compounds would differ pharmacologically just as they differed chemically. A pharmacologic difference was strongly suggested by our early observations on the effect of ticrynafen on diuretic and natriuretic activity in the mongrel dog. Ticrynafen, administered to the hydrated dog at doses of 12.5, 25 and 50 mg/kg orally, was compared, in an incomplete l a t i n block design, with ethacrynic acid at oral doses of 3.1, 6.25 and 12.5 mg/kg. Urine was collected from these dogs for 5 hours and then for an additional 19 hours, for a total of 24 hours. Compared for diuretic and natriuretic effectiveness at 5 hours, ticrynafen was l/26th and l/23rd as active, respectively, as ethacrynic acid. However, after 24 hours collection, ticrynafen was 1 / l l t h as active a diuretic and l/14th as active a natriuretic agent as ethacrynic 0-8412-0464-0/78/47-083-084$05.00/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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acid. These data demonstra id that not only d i d the two compounds d i f f e r i n d u r a t i o n o. a c t i o n , with t i c r y n a f e n being an e f f e c t i v e d i u r e t i c and n a t r i u r e t i c over a longer time span than e t h a c r y n i c a c i d , but a l s o suggested t h a t the n a t r i u r e t i c potency of t i c r y n a f e n d i f f e r e d from e t h a c r y n i c a c i d . Since the response to a d i u r e t i c i s p r i n c i p a l l y determined by the l o c u s i n the nephron where the drug e x e r t s i t s i n h i b i t o r y e f f e c t on sodium r e a b s o r p t i o n ( 7 ) , we attempted to e s t a b l i s h the s i t e of r e n a l a c t i o n of t i c r y n a f e n . There are f o u r major s i t e s of d i u r e t i c a c t i v i t y i n the r e n a l tubule: a proximal s i t e ; two s i t e s , medullary and c o r t i c a l , i n the t h i c k ascending limb of the loop of Henle; and a f a r d i s t a l s o - c a l l e d sodium-potassium exchange s i t e . At one time or another, p r a c t i c a l l y every s i t e i n the tubule has been claimed as the a c t i v e s i t e f o r every d i u r e t i c (8). But, i n r e c e n t years i t has been accepted that the p a t t e r n of e l e c t r o l y t e e x c r e t i o n c l e a r a n c e i n hydrated an t e r i z e the s i t e o f d i u r e t i c a c t i v i t y o f a compound (J). A d u l t female mongrel dogs were hydrated by g i v i n g an o r a l water load o f 500 ml tap water t h i r t y minutes p r i o r to i n i t i a t i o n of standard r e n a l clearance procedure. Dogs were i n f u s e d i n t r a venously at 3 ml/minute throughout the course o f the experiment with a k% mannitol-phosphate b u f f e r s o l u t i o n , pH 7.4 (.9). Glomerular f i l t r a t i o n rate was determined by the clearance o f c r e a t i n i n e and e f f e c t i v e r e n a l plasma flow by the clearance of p-aminohippurate. T i c r y n a f e n , administered i n t r a v e n o u s l y to f o u r hydrated mongrel dogs, caused a moderate n a t r i u r e s i s , k a l i u r e s i s and c h l o r u r e s i s with only a nominal i n c r e a s e i n u r i n e volume. The s m a l l change i n urine flow r a t e above c o n t r o l (Table I) suggested that t i c r y n a f e n had l i t t l e or no proximal a c t i v i t y . In t h i s , t i c r y n a f e n d i f f e r e d from h y d r o c h l o r o t h i a z i d e , furosemide and acetazolamide. A l l of these compounds i n c r e a s e d sodium e x c r e t i o n which r e s u l t e d i n a p o s i t i v e osmolal clearance above c o n t r o l s (n = 6 ) . However, only acetazolamide caused a p o s i t i v e f r e e water c l e a r a n c e . These data suggested that t i c r y n a f e n was a c t i v e i n the t h i c k ascending limb of the loop of Henle. TABLE I DIURETIC EFFECTIVENESS IN HYDRATED DOGS Diuresis* ml/min

Drug

Sodium* FE

osm* ml/min

ml/min

+ 1.4 +14.2 + 4.4 + 3-8

+ -

t Acetazolamide Furosemide Hydrochlorothiazide T i c r y n a f e n (n = 4) * change from

3-2 10.0 1.8 O.2

3-3 15.6 5.3 6.3

1.8 4.2 2.5 3.6

control

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Adult, female mongrel dogs and dalmation coach hounds were f a s t e d overnight. Using a constant r a t e i n f u s i o n pump, the dogs were i n f u s e d i n t r a v e n o u s l y a t O.5 ml/minute throughout the course of the experiment with a 4*ί mannitol-phosphate b u f f e r pH 7-4. The same standard r e n a l clearance procedures were followed except t h a t u r i n e c o l l e c t i o n i n t e r v a l s were 20 minutes r a t h e r than the 10 minute c o l l e c t i o n periods used i n the hydrated dogs. In the hydropenic dogs; however, t i c r y n a f e n i s r e a d i l y d i s t i n g u i s h e d from furosemide by i t s l a c k o f a s i g n i f i c a n t a f f e c t upon T H 0 , the s o l u t e f r e e water o f r e a b s o r p t i o n . T i c r y n a f e n , administered i n t r a v e n o u s l y a t 15 mg/kg to e i g h t hydropenic dogs increased u r i n e flow r a t e above that o f the c o n t r o l s (Table I I ) i n the p r o t o c o l . Again, the n a t r i u r e t i c e f f e c t i v e n e s s o f t i c r y n a f e n was e q u i v a l e n t to that o f h y d r o c h l o r o t h i a z i d e , and again i t i n c r e a s e d osmolal c l e a r a n c e . However, the modest change on f r e e water o f reabsorption (T H 0) strongl c o r t i c a l d i l u t i n g segmen c

2

c

2

TABLE I I DIURETIC EFFECTIVENESS IN HYDROPENIC DOGS

Drug

Diuresis ml/min

Sodium FE

C

osm ml/min

T H ( ml/mi]

+ + + +

+ + +

C

% Acetazolamide Furosemide Hydrochlorothiazide T i c r y n a f e n (n = 8)

1.8 4.0 O.9 1.5

3.4 5.9 3.0 3-2

2.5 3.6 1.7 1.6

O.7 O.4 O.8 O.1

S i m i l a r data have been obtained i n the hydrated and dehy­ drated normal male volunteer (10). T i c r y n a f e n a t 500 o r 1000 mg had no e f f e c t upon u r i n e flow r a t e i n the hydrated v o l u n t e e r s (Table I I I ) , suggesting a l a c k o f proximal a c t i v i t y . T i c r y n a f e n , given o r a l l y to hydrated s u b j e c t s , increased sodium e x c r e t i o n and osmolal clearance but decreased f r e e water c l e a r a n c e . TABLE I I I DIURETIC EFFECTIVENESS IN HYDRATED NORMAL VOLUNTEERS

Dose

Diuresis ml/min

Sodium FE

C

osm ml/min

C

H 0 ml/min

% Control 500 mg (n = 3)

16.0 16.1

1.3 4.3

3.4 7.1

12.8 8.9

Control 1000 mg (n = 2)

15.0 14.7

1.0 5.7

3.3 7.7

11.8 6.9

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In hydropenic v o l u n t e e r s , t i c r y n a f e n , a t 500 or 1000 mg o r a l l y , a l s o increased sodium e x c r e t i o n and osmolal c l e a r a n c e . U n l i k e f r e e water clearance however, t u b u l a r r e a b s o r p t i o n of s o l u t e f r e e water rose and was e i t h e r equal to or exceeded that of the c o n t r o l s (Table I V ) . T h i s o b s e r v a t i o n , along with the o b s e r v a t i o n i n both hydrated and hydropenic normal v o l u n t e e r s , that only 4 to 6% of the f i l t e r e d sodium load was excreted, c l e a r l y p l a c e the s i t e of d i u r e t i c a c t i v i t y of t i c r y n a f e n i n the c o r t i c a l d i l u t i n g segment of the d i s t a l tubule (10). TABLE IV DIURETIC EFFECTIVENESS IN HYDROPENIC NORMAL VOLUNTEERS C

T H 0 ml/min

Diuresis ml/min

Sodium FE

Control

O.9

O.9

3.0

2.1

500 mg (n = 3)

3.3

4.6

6.4

3.1

Control

O.9

1.1

3.1

2.2

1000 mg (n = 2)

4.7

6.1

6.9

2.2

Dose

'-osm ml/mi

2

The congruence of r e s u l t s of a c t i v i t y of t i c r y n a f e n i n hydrated and hydropenic man and dog upon f r e e water clearance and the s o l u t e f r e e water of r e a b s o r p t i o n d e l i n e a t e i t s s i t e of a c t i o n (Table V ) . Proximal, loop and d i s t a l d i u r e t i c s a d m i n i s t e r ­ ed to hydrated dogs increased sodium e x c r e t i o n and osmolal clearance. Based on the p h y s i o l o g i c p r i n c i p l e s o u t l i n e d by Goldberg e t a l . (11), f r e e water c l e a r a n c e (^Έ 0) was i n c r e a s e d only by acetazolamide, a p r o x i m a l l y a c t i v e d i u r e t i c (12) and was decreased by loop and d i s t a l l y a c t i n g d i u r e t i c s . But i n hydropenic animals, the d i s t a l l y a c t i v e compounds, h y d r o c h l o r o ­ t h i a z i d e and t i c r y n a f e n caused a s l i g h t i n c r e a s e or no change i n T H20, c l e a r l y a d i f f e r e n t response than that obtained with furosemide (13), a "loop-agent". 2

C

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TABLE V SUMMARY OF THE EFFECTS OF DIURETICS UPON RENAL CONCENTRATING AND DILUTING MECHANISMS Experimental Procedure S i t e of A c t i o n

Hydrated %>0

Hydropenic T H 0 c

2

Proximal Tubule Acetazolamide Loop of Henle Furosemide D i s t a l Tubule Ticrynafen To determine the p a t t e r electrolyt dog, t i c r y n a f e n was administered i n t r a v e n o u s l y , i n s i n g l e acute doses, to the phosphate-mannitol i n f u s e d dog. At intravenous doses of O.05 to 15 mg/kg, the compound enhanced u r i n e volume and caused e x c r e t i o n of sodium, potassium and c h l o r i d e . The maximum n a t r i u r e t i c response occurred promptly w i t h i n the f i r s t 10 minutes a f t e r completion of the i n j e c t i o n , and as the dose was increased there was a g r e a t e r d u r a t i o n of n a t r i u r e s i s (Table V I ) . TABLE VI ELECTROLYTE EXCRETION FOLLOWING ADMINISTRATION OF TICRYNAFEN TO THE PHOSPHATE-MANNITOL INFUSED DOG I. V. Dose mg/kg

Placebo O.05 O.5 2.5 5.0 15.0

No of Observations

Maximal FË Na %

10 3 6 1 4 3

2.09 2.93 3.91 7.40 10.05 11.60

T o t a l Cumulative E x c r e t i o n mEq During F i r s t Hour Na Κ CI 5.29 9.39 7.02 12.96 19.31 33.85

1.49 1.36 1.13 2.80 2.09 4.58

2.64 6.33 4.00 11.61 15.46 31.80

When compared s t a t i s t i c a l l y w i t h placebo, t i c r y n a f e n caused a s i g n i f i c a n t n a t r i u r e t i c response at O.5 mg/kg I.V. which increased i n a l i n e a r f a s h i o n w i t h dose, reaching a maximum e f f e c t w i t h e x c r e t i o n of about 12% of the f i l t e r e d sodium load at 15 mg/kg. The e x c r e t i o n of c h l o r i d e p a r a l l e l s that of sodium but i s somewhat reduced due to the presence of phosphate i n the infusion-medium, suggesting that t i c r y n a f e n i s a s a l u r e t i c

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agent. A s i g n i f i c a n t increase i n potassium e x c r e t i o n occurred at a dose o f 2.5 mg/kg, f i v e times the minimum e f f e c t i v e n a t r i u r e t i c dose. This observation makes i t u n l i k e l y that t i c r y n a f e n has an i n h i b i t o r y a f f e c t upon the sodium/potassium exchange s i t e . During maximal n a t r i u r e s i s i n man, f o l l o w i n g a d m i n i s t r a t i o n of 500 mg t i c r y n a f e n , average sodium e x c r e t i o n i n c r e a s e d 463 yEq/ min., while potassium e x c r e t i o n increased only 16 μΕα/min. Clearance r a t i o of calcium to i n u l i n increased by approximately 1% and clearance r a t i o o f magnesium 2.6j&. Bicarbonate and phosphorus e x c r e t i o n s were not s i g n i f i c a n t l y changed, and u r i n a r y pH c o n s i s t e n t l y decreased. The acute a d m i n i s t r a t i o n o f t i c r y n a f e n d i d not r e s u l t i n any s i g n i f i c a n t change i n GFR as determined by i n u l i n c l e a r a n c e . T h i s e l e c t r o l y t e p a t t e r n again supports a c o n c l u s i o n of a l a c k of a c t i v i t y of t i c r y n a f e n i n the proximal tubule, while th similarit f electrolyt t "thiazide-like" diuretic c o r t i c a l d i l u t i n g segmen As a r e s u l t of these observations, we have concluded that h y d r o c h l o r o t h i a z i d e and t i c r y n a f e n , two compounds with markedly d i f f e r i n g chemical s t r u c t u r e , produce a s i m i l a r p a t t e r n o f e l e c t r o l y t e response due to t h e i r a c t i v i t y at the same s i t e i n the kidney tubule. While d i u r e t i c s of markedly d i f f e r i n g chemical s t r u c t u r e have not been recognized p r e v i o u s l y to be a c t i v e a t a c o r t i c a l d i l u t i n g s i t e , compounds of widely d i f f e r i n g s t r u c t u r e - e t h a c r y n i c a c i d , bumetanide, furosemide and m e r s a l y l - have been recognized to be a c t i v e a t a medullary d i l u t i n g s i t e on the t h i c k ascending limb o f Henle's loop. Thus, s p e c i f i c s t r u c t u r e - f u n c t i o n r e l a ­ t i o n s h i p s do not appear to e x i s t among d i u r e t i c s except i n s o f a r as they are organic a c i d s . But no one, I b e l i e v e , would c l a i m that a l l organic a c i d s are d i u r e t i c s , and conversely two d i u ­ r e t i c s , triamterene and a m i l o r i d e , a c t i v e at the f a r d i s t a l sodium/potassium exchange s i t e c l e a r l y cannot be c l a s s i f i e d as organic a c i d s . However, molecular s t r u c t u r e s determining p a r t i c i p a t i o n i n the organic a c i d t u b u l a r t r a n s p o r t system are c l e a r l y p e r t i n e n t . The d i u r e t i c e f f e c t of m e r s a l y l (14) and e t h a c r y n i c a c i d (15) appears to be due to i n h i b i t i o n by these drugs i n the t u b u l a r lumen of a c t i v e c h l o r i d e t r a n s p o r t i n the t h i c k ascending limb o f Henle's loop. S i m i l a r data are a v a i l a b l e f o r furosemide i n v i t r o ( 1 6 ) and i n man (1J); the d i u r e t i c response i s determined by the amount of furosemide that reaches the r e n a l tubule r a t h e r than the drug l e v e l i n plasma (18). S i m i l a r l y , i n the dog, i t has been found that those compounds which, administered i n s u f f i c i e n t dose, i n h i b i t r e n a l t r a n s p o r t of t i c r y n a f e n , a l s o reduce t i c r y n a f e n n a t r i u r e s i s (19). To add to the complexities f a c i n g the m e d i c i n a l chemist s y n t h e s i z i n g a new d i u r e t i c , i s the observation that t i c r y n a f e n has a s i g n i f i c a n t u r i c o s u r i c a c t i v i t y . That an organic a c i d should have u r i c o s u r i c a c t i v i t y i s not t o t a l l y unexpected. Any f

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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l i s t o f u r i c o s u r i c agents, from acetoheximide to zoxazolamine w i l l e a s i l y i n c l u d e 30 compounds o f widely d i f f e r i n g chemical s t r u c t u r e , the majority o f which are organic a c i d s . However, weak organic bases such as zoxazolamine are a l s o u r i c o s u r i c . What i s unexpected i s that o r a l l y a c t i v e d i u r e t i c s which should increase u r i c a c i d e x c r e t i o n , g e n e r a l l y cause a s i g n i f i c a n t u r i c a c i d r e t e n t i o n , p o s s i b l y due to t h e i r c o n t r a c t i o n of e x t r a ­ c e l l u l a r f l u i d volume (20) and/or i n h i b i t i o n of urate s e c r e t i o n . In the phosphate-mannitol infused mongrel dog, t i c r y n a f e n at doses o f 0.5 to 15 mg/kg intravenously, i s n a t r i u r e t i c and u r i c o s u r i c (Table V I I ) . Glomerular f i l t r a t i o n r a t e was deter­ mined by the clearance o f H - i n u l i n , and p-aminohippurate was omitted from the i n f u s i o n s o l u t i o n . The dogs were not urate loaded. Under these c o n d i t i o n s , t i c r y n a f e n was found to be n a t r i u r e t i c and u r i c o s u r i c potency to probenecid. o r i g i n a l r e p o r t s o f u r i c o s u r i a induced i n the mongrel dog, reported by Beyer et a l . (21) and by Sougin-Mibashan and Horwitz (22). ~~ TABLE VII DIURETIC AND URICOSURIC ACTIVITY IN THE MONGREL DOG

Compound

Sodium* FE

°Ur* ml/min

i Probenecid 10 mg/kg I.V. Hydrochlorothiazide 0.5 mg/kg +0.5 mg/kg/hr Ticrynafen 1.5 mg/kg +1.5 mg/kg/hr 4 mg/kg I.V. 10 mg/kg I.V. 15 mg/kg I.V.

Urate* FE %

+ 16.2

+ 31

+ 5.3

- 2.3

- 1

2.1 4.2 4.7 8.5

+ 8.7 + 7.5 + 9.8 +12.6

0

+ + + +

+ + + +

18 17 25 22

* change from c o n t r o l η Ur = clearance o f urate In man, t i c r y n a f e n i s markedly u r i c o s u r i c (Table V I I I ) , the f r a c t i o n a l e x c r e t i o n o f urate r i s i n g to a maximum i n one volun­ teer o f 75% o f the f i l t e r e d l o a d . As a consequence o f the u r i c o s u r i a , serum urate l e v e l s and, consequently, the f i l t e r e d load o f urate to the kidney i s sharply reduced. I n c o n t r a s t , the a b i l i t y o f the o r a l l y a c t i v e d i u r e t i c s , e s p e c i a l l y the t h i a z i d e s and furosemide, to r e t a i n u r i c a c i d and increase the f i l t e r e d urate l o a d i s w e l l recognized (23).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

6.

MAASS

ET AL.

91

Ticrynafen TABLE VIII

URICOSURIC ACTIVITY OF TICRYNAFEN IN HYDRATED NORMAL MALE VOLUNTEERS υ.. V Dose c c Ur Volunteer mg Ur/ I n χ 100 pg/min S-82

S-83

C Ε

500

634 2558

8.8 38.6

C Ε

500

600 2680

12.3 63.3

S-84

C Ε

S-127

C Ε

mmmm

1000

475

7.5

680 3173

7.7 58.8

Although researchers i n the f i e l d o f urate t r a n s p o r t do not agree on the r e n a l tubular s i t e o f urate r e a b s o r p t i o n , a proximal s i t e o f urate t r a n s p o r t i s considered p o s s i b l e . S u l f i n p y r a z o n e , probenecid and a s p i r i n are secreted by an organic a c i d s e c r e t o r y c a r r i e r l o c a t e d i n the proximal tubule (24). Diamond and M e i s e l (25) have demonstrated that probenecid s e c r e t i o n i s r e q u i r e d f o r i t s u r i c o s u r i c e f f e c t , and they have proposed that competition f o r b i n d i n g a t a u r a t e - r e a b s o r p t i v e s i t e on the tubular luminal surface may account f o r the e f f e c t o f probenecid (26). In the mongrel dog, competition with t i c r y n a f e n s e c r e t i o n reduced the u r i c o s u r i c a c t i v i t y o f t i c r y n a f e n as i t reduced the n a t r i u r e t i c a c t i v i t y o f the drug. Thus, i n c r e a s i n g l y a case may be made f o r the proximal t r a n s p o r t o f d i u r e t i c s t o a c t from the tubular lumen (19). I t i s a challenge to the medicinal chemist t o f i t h i s newly synthesized molecules t o t h i s r e n a l t r a n s p o r t system. In summary, the data obtained i n t h i s study o f t i c r y n a f e n c l e a r l y i n d i c a t e that m o d i f i c a t i o n o f chemical s t r u c t u r e s t o o b t a i n new d i u r e t i c s r e q u i r e s the extensive p a r t i c i p a t i o n o f the p h y s i o l o g i s t . Literature Cited 1. Burg, M. and Stoner, L., Ann. Rev. Physiol. (1976), 38, 37-45. 2. Goldberg, M . , McCurdy, D. K . , F o l t z , E . L . and Bluemle, L. W., J. C l i n . Invest. (1964), 43, 201-216. 3. Puschett, J. B. and Goldberg, M . , J. Lab. C l i n . Med. (1968), 71, 666-676. 4. Jayakumar, S. and Puschett, J. B . , J. Pharm. Exptl. Therap. (1977), 201, 251-258. 5. Burg, Μ., "The Mechanism of Action of Diuretics i n Renal

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Tubules." Recent Advances Renal Physiol. Pharm., E d . , L . G. Wesson and G. M. F a n e l l i , university Park Press, (1974). 6. Beyer, K. H . , Baer, J. E., Michaelson, J. K. and Russo, H. F., J. Pharm. Exptl. Therap. (1965), 147, 1-22. 7. Seldin, D. W., Eknoyan, G . , Suki, W. N. and Rector, F . C., Ann. Ν. Y. Acad. S c i . (1966), 139, 328-343. 8. Clapp, J. R. and Robinson, R. R., Am. J. Physiol. (1968), 215, 228-235. 9. Baer, J. E., Michaelson, J. K . , McKinstry, D. N. and Beyer, K. H . , Proc. Soc. Exptl. B i o l . Med. (1964), 115, 87-90. 10. Stote, R. M . , Maass, A. R. C h e r r i l l , D. Α . , Beg, M. M. A. and Alexander, F., J. Pharmacol. C l i n . (Special Issue 1976), 19-27. 11. Goldberg, M . , McCurdy, D . , Foltz, E . and Bluemle, L., J. C l i n . Invest. (1964), 43, 201-216. 12. Buckalew, V. Μ., Walker B R. Puschett J. B and Goldberg, M . , J. C l i n . Invest 13. Seldin, D. W., Eknoyan Ann. Ν. Y. Acad. S c i . (1966), 139, 328-343. 14. Burg, M. and Green, N . , Kidney Int. (1973), 4, 245-251. 15. Burg, M. and Green, Ν . , Kidney Int. (1973), 4, 301-306. 16. Burg, M . , Stoner, L., Cardinal, J. and Green, N . , Am. J. Physiol. (1973), 225, 119-124. 17. Homeida, H . , Roberts, C. and Branch, R. Α . , C l i n . Pharm. Therap. (1977), 22, 402-409. 18. Honari, J., B l a i r , A. D. and Cutler, R. E., C l i n . Pharm. Therap. (1977), 22, 395-401. 19. Maass, A. R. and Snow, I . B. To be published. 20. Steele, T. H. and Oppenheimer, S., Am. J. Physiol. (1969), 47, 564-574. 21. Beyer, Κ. Η . , Russo, H. F., T i l l s o n , Ε. Κ., M i l l e r , Α. Κ., Verwey, W. F., Gass, S. R., Am. J. Physiol. (1951), 166, 625-640. 22. Sougin-Mibashan, R. and Horwitz, M . , Lancet (1955), 1, 1191-1197. 23. Wyngaarten, J. B. and Kelley, W. N . , "Drug-Induced Hyper­ uricemia and Gout" i n Gout and Hyperuricemia, 369-370, Grune & Stratton (1976). 24. Tune, Β. Μ., Burg, M. B. and Patlak, C. S., Am. J. Physiol. (1969), 217, 1057-1063. 25. Meisel, A. D. and Diamond, H. S., Arth. Rheum. (1977), 20, 128. 26. Diamond, H. S. and Meisel, A. D . , C l i n . S c i . Mol. Med. (1977), 53, 133-139. RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7 2-Aminomethylphenols: A New Class of Saluretic Agents R. L . SMITH, E . M . SCHULTZ, G. E . STOKKER, and E . J. CRAGOE, JR. Merck Sharp and Dohme Research Laboratories, West Point, PA 19486

A continuing searc fo i laboratorie by screening carefully r e t i c activity i n rats and dogs led to the discovery of 2-amino methyl-3,4,6-trichlorophenol (Ia). The unusual structural fea­ tures, attractive electrolyte excretion profile and saluretic potency of compound Ia relative to those of known diuretics (see Table I for an activity comparison) provided impetus for an extensive synthetic program. The data obtained f a c i l i t a t e d the delineation of the structure-activity relationships for a variety of 2-aminomethylphenols and culminated i n the development of 2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride (MK-447). This compound was found to be a potent, high-ceiling salidiuretic agent with adjunctive antihypertensive and a n t i ­ -inflammatory properties, and i t is currently undergoing c l i n i c a l evaluation. This report i s a preliminary account (1) of the highlights of our research on this new class of saluretic agents.

TABLE I.

Relative Saluretic A c t i v i t i e s Species

Compound

Rat

la Hydrochlorothiazide Furosemide Ethacrynic Acid a

3

(Admin. Route)

(ρ.ο.)

Dog

(i.v.)

3

4

2

2

3

5

0

5

Presented as scores; assay p r o t o c o l s are d e s c r i b e d in the t e x t , and s c o r i n g criteria are presented i n Tables II and III.

0-8412-0464-0/78/47-083-093$08.25/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

94

AGENTS

SALIDIURETIC ACTIVITY ASSAYS The structure-activity relationships (vide infra) in this study were determined by evaluating each synthetic compound for salidiuretic activity i n two animal species, the rat and the dog. A brief description of each assay protocol is given below along with the system used to score the biological results. Oral Rat Assay - Female rats (Charles River, 150-170 g), housed in metabolism cages in groups of three rats per cage, were maintained overnight on a sugar diet with water ad libitum. At the time of the test, each animal was given the test compound orally either as a solution or suspension in 5 ml of water. Urine was collected over the 0-5 hr interval in graduated cylinders and subsequently analyzed for sodium, potassium and chloride content by standard methodology The saluretic response was scored from 0 to 6 according to the natriureti TABLE I I .

Scoring System f o r O r a l Rat Assay +

yEq Na /cage, dose i n mg/kg Score

1

5

50

81

0.3

0.3

0.4

0.7

0.9 1.5

16.5

0

+ 1

0.3

0.5

1.2

2

0.4

0.6

1.8

2.4

3

0.5

1.4

2.4

2.9

4

1.4

2.7

3.3

5

0.7

2.6

3.3

3.6

6

2.1

3.6

4.2

3.9

Intravenous Dog Assay - Conditioned female mongrel dogs, weighing approximately 20 kg i n the postabsorptive s t a t e , were starved overnight and then given 500 ml o f water o r a l l y 1 h r before i n d u c t i o n o f a n e s t h e s i a with sodium p e n t o b a r b i t a l (30 mg/kg, i . v . ) . A f t e r inducing anesthesia, each dog was prepared with an i n d w e l l i n g bladder c a t h e t e r and primed with c r e a t i n i n e Ο g as a 10% s o l u t i o n i n water) administered s.c. i n m u l t i p l e i n j e c t i o n s i t e s . To ensure uniform hydration and u r i n e pro­ duction, 1.5 ml/kg o f an isoosmotic pH 7·4 phosphate b u f f e r s o l u t i o n (20 mg phosphate/kg) was given i . v . as a priming i n j e c ­ t i o n p r i o r to i n i t i a t i o n o f clearance s t u d i e s and 3 ml/min o f an isoosmotic pH 7 Λ b u f f e r c o n t a i n i n g k% mannitol (6.9 mg phosphate/min) was i n f u s e d during the experiment. A t the s t a r t o f

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7.

SMITH ET AL. 2~Aminomethylphenols

95

timed c l e a r a n c e s , the u r i n a r y bladder was emptied and r e p l i c a t e 15-min u r i n e c o l l e c t i o n s were made with venous blood samples being drawn at the midpoint o f each p e r i o d . F o l l o w i n g t h i s c o n t r o l phase, the t e s t compound was administered i . v . s t a t ( i . e . , over a 5 min period) a t 5 mg/kg. Urine was c o l l e c t e d over r e p l i c a t e 15 min p e r i o d s f o r 2 hr and subsequently assayed f o r e l e c t r o l y t e content by standard methodology. The average r a t e o f n a t r i u r e s i s determined f o r the two highest consecutive 15 min c o l l e c t i o n periods was used to score the s a l u r e t i c response from 0 to 5 on the b a s i s o f the sodium e x c r e t i o n r a t e s i n d i c a t e d i n Table I I I .

TABLE I I I .

Scoring System f o r Intravenous

yEq Na /min Score

I.V.

Dog

Assay

at 5 mg/kg Stat

0

0-99

+

a c t i v e above 5 mg/kg

1

100-399

2

400-599

3

600-799

4

800-899

5

900

STRUCTURE-ACTIVITY RELATIONSHIPS The i n i t i a l phase o f t h i s i n v e s t i g a t i o n was d i r e c t e d toward determination o f the b i o l o g i c a l consequences r e s u l t i n g from r e o r i e n t a t i o n and s t r u c t u r a l m o d i f i c a t i o n o f the hydroxyl ( p h e n o l i c ) and aminomethyl groups i n compound l a . As i s shown i n Table IV, o r i e n t a t i o n o f these f u n c t i o n a l groups i n e i t h e r a meta (Compound l b ) or para (Compound I c ) r e l a t i o n s h i p with concomitant p o s i t i o n a l interchange o f the aminomethyl moiety with e i t h e r the 3- or 4-chloro s u b s t i t u e n t r e s u l t e d i n a b l a t i o n o f a c t i v i t y . Hence, these r e s u l t s e s t a b l i s h e d the importance o f m a i n t a i n i n g an ortho r e l a t i o n s h i p between the hydroxyl and aminomethyl groups.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

TABLE IV.

Orientation Effects

OH

S a l u r e t i c Score

x

Compd. Ia

x

2

CH NH 2

b

CI

c

CI

3

CI

2

x

4

CI

Rat

Dog

3

4

0

0

The e f f e c t s o f N - s u b s t i t u t i o n on s a l u r e t i c a c t i v i t y were explored next and a r e tabulated f o r a r e p r e s e n t a t i v e s e r i e s o f s t r u c t u r e s i n Table V· As the data i n d i c a t e , a l k y l a t i o n o f the amino group proved t o be detrimental t o a c t i v i t y as d i d a c y l a t i o n , with exception o f N - t r i f l u o r o a c e t y l a t i o n . In the l a t t e r case, the a c t i v i t y e l i c i t e d upon o r a l a d m i n i s t r a t i o n o f compound I l g i n the rat most l i k e l y r e s u l t e d from i t s i n v i v o h y d r o l y s i s t o the a c t i v e precursor, compound l a . Indeed, the observed f a c i l e conversion o f t r i f l u o r o a c e t a m i d e I l g t o amine I a under m i l d h y d r o l y t i c c o n d i ­ t i o n s (e.g., 20°C, pH 8, 30 min) i n v i t r o i s c o n s i s t e n t with t h i s viewpoint. Meadow, e t a l (2, 3) have reported some o f the b i o l o g i c a l a c t i v i t i e s d i s p l a y e d by a s e r i e s o f t e r t i a r y amines o f general formula I I I ; compound I l i a was i d e n t i f i e d as the most a c t i v e d i u r e t i c member o f the s e r i e s . In our l a b o r a t o r i e s , compound 5H I l i a was found to be i n a c t i v e i n both the r a t and dog d i u r e t i c :H N(CH CHR) Y assays under a v a r i e t y o f t e s t conditions. This r e s u l t i s i n t e r e s t i n g i n view o f the a c t i v i t y , a l b e i t weak, d i s p l a y e d by t e r t i a r y amine l i e . A l t e r a t i o n o f the amino­ I l i a , R=H; Y=0; methyl group, e.g., by a - s u b s t i t u ­ X =CH CH=CH ; t i o n (Compound IVa), homologation (Compound IVb) o r simultaneous X =0CH α-substitution and homologation (Compound I V c ) , l e d t o s u b s t a n t i a l diminution i n a c t i v i t y as demonstrated i n Table V I . Furthermore,

Ο

0

0

0

4

2

2

6

3

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

SMITH ET AL.

2-Aminomethylphenols

TABLE V Effects of N-Substitution

(II)

Cl

Salureti

Compd

R

lia

NHCH

b

N

(

C

Rat

1

3

V 2

N(CH CH ) 0

d

N(CH )CH -I^

e

2

2

2

3

°

2

c

2

NHCHO

+

1

1

1

0

0 0

f

NHC0CH C1

1

g

NHCOCF

3

h

2

3

NHCOCH NH 2

Dog

2

+

1

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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the corresponding s a l i c y l a l c o h o l (Compound IVd), s a l i c y l a l d e h y d e (Compound IVe) and s a l i c y l i c a c i d (Compound I V f ) analogs o f compound I a were devoid o f demonstrable s a l u r e t i c a c t i v i t y . TABLE V I .

E f f e c t s o f Aminomethyl Group A l t e r a t i o n

OH .χ

2

(IV) ^Cl S a l u r e t i c Score

CI

x

Compd. IVa

2

Rat

Dog

1

^1

0

1

CH(CH

b

CH CH NH

c

CH(OH)CH NH

2

2

2

2

2

d

CH OH 2

0

0

e

CHO

0

0

f

C0 H

0

0

2

To complete the p r e l i m i n a r y SAR s t u d i e s , the e f f e c t s o f hydroxyl group m o d i f i c a t i o n were determined; the r e s u l t s a r e presented i n Table V I I . As the data i n d i c a t e , both O - a l k y l a t i o n (e.g., as i n compounds Va-c) and replacement o f OH by NH r e s u l t e d i n s t r u c t u r e s with marginal s a l u r e t i c a c t i v i t y . These r e s u l t s coupled with those presented p r e v i o u s l y (vide supra) i n d i c a t e d that: (a) the ortho o r i e n t a t i o n o f the hydroxyl and aminomethyl groups ( i . e . , as i n s a l i c y l a m i n e ) should be maintained, and that (b) these two f u n c t i o n a l groups must remain u n s u b s t i t u t e d . Ac­ c o r d i n g l y , the e f f e c t s o f nuclear s u b s t i t u t i o n , both s i n g u l a r l y and m u l t i p l y , were i n v e s t i g a t e d i n a systematic manner as an approach to improving s a l u r e t i c a c t i v i t y . 2

Data f o r a r e p r e s e n t a t i v e s e r i e s o f monosubstituted s a l i c y 1 amines (Compound VI) bearing s u b s t i t u e n t s i n the 4 - p o s i t i o n are shown i n Table V I I I . Although s a l i c y l a m i n e i t s e l f (Compound IVa) i s devoid o f demonstrable s a l i d i u r e t i c a c t i v i t y , i n t r o d u c t i o n o f a c h l o r o group i n the 4 - p o s i t i o n (Compound VIb) imparts weak s a l u ­ r e t i c p r o p e r t i e s which a r e maintained upon replacement o f the c h l o r o group by lower a l k y l groups up t o and i n c l u d i n g a three carbon s t r a i g h t chain, with or without α-branching. However, i n t r o d u c t i o n o f the η-butyl group i n the 4 - p o s i t i o n i s not t o l ­ erated, s i n c e compound V l f d i d not e x h i b i t demonstrable a c t i v i t y i n e i t h e r the r a t o r the dog assay.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

SMITH ET AL. 2-Aminomethylphenols

TABLE V I I E f f e c t s of Phenol Group M o d i f i c a t i o n

Va

OCH

3

±

0

1

0

b

OCH C0 C H

c

OCH C0 H

+

d

NH

±

2

2

2

2

2

2

5

0

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

100 TABLE V I I I . OH

E f f e c t s of Nuclear

J

è

Monosubstitution

2 (VI)

S a l u r e t i c Score Compd. Via b c

x

4

Rat

Dog

H

0

0

Cl

1

1

C

d

C(CH )

e

CH(CH )C H

f

(CH ) CH

3

3

2

1

3

2

3

3

5

1

0

0

0

Appropriate nuclear d i s u b s t i t u t i o n l e d to marked enhancement of s a l u r e t i c a c t i v i t y as i n d i c a t e d by the r e s u l t s tabulated i n Table IX. The d i c h l o r o d e r i v a t i v e s (Compounds VIIa-c) i l l u s t r a t e the importance of the 4,6-çlisubstitution p a t t e r n , i . e . , V H b i s more a c t i v e than the other p o s i t i o n a l isomers. T h i s *observation, coupled with the information gained from the monosubstituted s e r i e s , prompted preparation o f compounds V I I d - i . Replacement o f the 4 - c h l o r o group i n compound V H b with lower a l k y l groups l e d to markedly improved a c t i v i t y which peaked i n potency with the i n t r o d u c t i o n of a 4 - ( l , l - d i m e t h y l e t h y l ) moiety, i . e . , compound Vlli. I n t e r e s t i n g l y , the 6 - c h l o r o , 6-bromo and 6 - i o d o d e r i v a t i v e s ( i . e . , compounds V l l i , V l l k and V I I I , r e s p e c t i v e l y ) e l i c i t marked s a l i d i u r e t i c responses, whereas, the 6 - f l u o r o analog (Compound V I I j ) i s considerably l e s s a c t i v e . As w i l l be discussed subsequently, the pronounced a n t i h y p e r t e n s i v e p r o p e r t i e s o f M K - 4 4 7 (Compound VIII) served to d i s t i n g u i s h i t from a subseries o f nearly equipotent s a l u r e t i c agents which emerged during the course o f t h i s i n v e s t i g a t i o n . F i n a l l y , i t should be noted that compound Vllm, the primary amine analog of compound I l i a (vide supra), d i s p l a y e d good a c t i v i t y i n both r a t s and dogs. The i n f l u e n c e s of nuclear t r i s u b s t i t u t i o n on s a l u r e t i c a c t i v i t y are presented i n Table X f o r a group o f compounds o f the general s t r u c t u r e V I I I . I t i s noteworthy that s h i f t i n g the 3 - c h l o r o s u b s t i t u e n t i n compound Ia to the 5 - p o s i t i o n ( i . e . , to a f f o r d compound V i l l a ) r e s u l t e d i n s l i g h t l y improved a c t i v i t y i n the dog; whereas, movement o f the 6 - c h l o r o group i n Ia to the

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7.

SMITH E T AL. 2-Aminomethylphenob

101

TABLE IX

E f f e c t s o f Nuclear D i s u b s t i t u t i o n

2 (VII)

S a l u r e t i c Score

A

Compd

x

3

Cl

Cl

H

H

1

1

b

H

Cl

H

Cl

2

+

c

Cl

H

H

Cl

0

1

d

H

CH

3

H

Cl

2

1

e

H

C H

5

H

Cl

4

5

f

H

(CH ) CH

3

H

Cl

4

5

g

H

CH CH(CH )

2

H

Cl

3

2

h

H

CH(CH )C H

5

H

Cl

5

4

i

H

C(CH )

3

H

Cl

6

5

j

H

C(CH )

3

H

F

4

5

k

H

C(CH )

3

H

Br

6

5

H

C(CH )

3

H

I

6

5

H

CH CH=CH

H

0CH

3

2

Vila

a

l m

2

2

2

2

3

3

2

3

3

3

3

2

2

3

Compound V I I I = MK-447

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

102

5 - p o s i t i o n ( i . e . , to form compound V H I b ) diminished a c t i v i t y i n the dog while m a r g i n a l l y enhancing a c t i v i t y i n the r a t . F u r t h e r ­ more, i n t r o d u c t i o n o f e l e c t r o n donating s u b s t i t u e n t s (e.g., methyl and methoxy groups) i n the 6 - p o s i t i o n , as i n compounds V H I f and V H I g , proved to reduce a c t i v i t y i n both t e s t s p e c i e s . TABLE X.

E f f e c t s o f Nuclear T r i s u b s t i t u t i o n

(VIII)

Compd.

Χ

3

x*

X

x

Rat

Dog

Ia

Cl

Cl

H

Cl

3

4

Villa

H

Cl

Cl

Cl

4

4

b

Cl

Cl

Cl

H

4

2

c

F

Cl

H

Cl

4

4

d

Cl

Cl

H

CF

3

4

2

H

Cl

3

2

2

1

1

1

1

0

e f

H

° 3

C H

CH

CH

3

3 3

H

H

° 3

g

Cl

Cl

H

0CH

h

OH

Cl

H

Cl

3

F i n a l l y , the data recorded i n Table XI i l l u s t r a t e the e f f e c t s o f nuclear t e t r a s u b s t i t u t i o n on a c t i v i t y . These data r e v e a l s e v e r a l i n t e r e s t i n g SAR trends. F i r s t , although r e p l a c e ­ ment o f the 3- and 5-chloro s u b s t i t u e n t s o f t e t r a c h l o r o d e r i v a t i v e IXa with methyl groups r e s u l t e d i n g r e a t l y enhanced a c t i v i t y i n the dog, most s u r p r i s i n g l y , the interchange o f c h l o r o and methyl s u b s t i t u e n t s ( i . e . , conversion o f Compound IXb t o compound IXc) t o t a l l y a b o l i s h e d a c t i v i t y . These r e s u l t s i n d i c a t e that both s t e r i c and e l e c t r o n i c e f f e c t s c o n t r i b u t e s u b s t a n t i a l l y i n determining the s a l u r e t i c e f f i c a c y o f these s t r u c t u r e s . Reinforcement f o r the importance o f these e f f e c t s i s provided by a c t i v i t y comparisons o f s t r u c t u r e s IXe with I X f and IXg with IXh. Diminution o f a c t i v i t y accompanies replacement

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7. SMITH ET AL.

2-Aminomethylphenols

103

of a chloro s u b s t i t u e n t with a methyl group ( e l e c t r o n i c e f f e c t ) i n the f i r s t instance and r e f l e c t s the r a t h e r s t r i n g e n t s t e r i c requirements f o r s u b s t i t u e n t s i n the 3 - p o s i t i o n i n the second i n s t a n c e . Secondly, the r e s u l t s tabulated f o r compounds IXb, I X i and IXk suggest t h a t , whereas the methyl groups i n the 3 - and 5 - p o s i t i o n s can be replaced with methoxy groups with maintenance of a c t i v i t y , t h e i r replacement with ethoxy moieties s u b s t a n t i a l l y reduces a c t i v i t y . The l a t t e r r e s u l t i s i n accord with the s t e r i c r e s t r a i n t s discussed above f o r s u b s t i t u e n t s i n the 3 - p o s i t i o n . Furthermore, i t should be noted that compounds IXg and I X i d i s p l a y s a l u r e t i c a c t i v i t i e s which are e s s e n t i a l l y o f the same magnitude as those o f the 4 - ( l , l - d i m e t h y l e t h y l ) - 6 - h a l o s a l i c y l a m i n e s ( i . e . , V l l i , V l l k and V I I I ) c i t e d e a r l i e r .

TABLE XI. JH

Effect

2

(IX)

x

3

S a l u r e t i c Score Compd. IXa b c

x

3

X

CI CH

CI CI

3

CI

CH

CH

3

e

CH

3

CH

3

f

CH

3

CH

3

g

CH

3

h

C H 2

i

0CH

Br

CH

4

1

CI

4

5 0

3

5

CI

CI

5

2

CH

CH

3

3

1

CI

5

5

CI

1

1 5

3

CI

5

CI

4

C H

3

Dog

0

CI

OCH

Rat

3

5

OC H

6

Br

C

j

3

CH

CI CH

CI

k

x

5

CI

3

d

2

x

4

3

H

2 5

3 0CH

3

CI

5

CI

0CH

3

Br

5

3

CI

OC H

5

CI

3

0

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

104

In summary, these SAR s t u d i e s i n d i c a t e that s a l u r e t i c a c ­ t i v i t y i s e l i c i t e d by 2-aminomethylphenols which a r e a p p r o p r i a t e l y s u b s t i t u t e d with a hydrogen, methyl or methoxy group i n the 3 - p o s i t i o n , a halo or lower a l k y l ( i . e . , < three carbon s t r a i g h t c h a i n , p r e f e r a b l y α-branched) moiety i n the 4 - p o s i t i o n , a hydrogen or lower a J k y l (or alkoxy) s u b s t i t u e n t i n the 5 - p o s i t i o n and a iodo, bromo o r c h l o r o group i n the 6 - p o s i t i o n , the optimal sub­ s t i t u e n t combinations and p a t t e r n s being determined by the c i t e d SARs. CHEMISTRY The two general s y n t h e t i c routes which were most f r e q u e n t l y used to prepare the s a l i c y l a m i n e d e r i v a t i v e s studied during the course o f t h i s i n v e s t i g a t i o n a r e summarized i n Scheme I The f i r s t route i n v o l v e s a c i d - c a t a l y z e appropriately substitute methylamide, RC0NHCH 0H (R=CH , OELC1, CCI-, CF or CgH ), t o g i v e an N - a c y l s a l i c y l a m i n e o f general formula XI. Subsequent hydrol y t i c N-deacylation o f compound XI Jfollowed by halogenation a f f o r d s the t a r g e t product. The second route i n v o l v e s the same steps but i n an a l t e r e d order, i . e . , halogenation o f phenol X t o give an o-halophenol o f general s t r u c t u r e XII followed by amido­ a l k y l a t i o n and h y d r o l y s i s . I n both sequences, the key amidomethylation step i s accomplished r e a d i l y v i a the Tscherniac-, Einhorn r e a c t i o n ( 4 ) . The choice o f the proper r e a c t i o n sequence, as w e l l as optimum c o n d i t i o n s ( i . e . , RCONHCH^OH, a c i d c a t a l y s t , r e a c t i o n medium, etc.) f o r i n t r o d u c i n g the aminomethyl moiety, i s governed p r i m a r i l y by^the^chemical nature and d i r e c t i n g i n f l u e n c e s o f the s u b s t i t u e n t s Χ , X and X . The importance o f s e l e c t i n g the proper a c i d c a t a l y s t and r e a c t i o n medium to c o n t r o l the a m i d o a l k y l a t i o n step i s demonstrated i n equation 1. 2

OH

3

NHC0CH Cl

CI XIII

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

o

7. SMITH ET AL.

2-Aminomethylphenoh

Scheme I

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

105

DIURETIC AGENTS

106

The d e s i r e d amide (Compound XIa) i s formed i n good y i e l d (6070%) when HOAc-H^SO^ (9:1) i s used, whereas, the use o f ^ S O ^ alone leads to extensive diamidomethylation (diamide X I I I ; , even when equimolar q u a n t i t i e s o f phenol Xa and N-hydroxymethylchloroacetamide are employed. A p p l i c a t i o n o f these general s y n t h e t i c routes t o the elabo­ r a t i o n o f MK-447 (5) from 4-(1,1-dimethylethyl)phenol (Compound Xb) i s shown i n Figure 1. The r e a c t i o n sequence, Xb >XIb > XIV >MK-447, i s the p r e f e r r e d s y n t h e t i c pathway f o r p r e p a r i n g large q u a n t i t i e s o f MK-447 and can be accomplished r o u t i n e l y i n 50% o v e r a l l y i e l d . Since the 6-iodo s u b s t i t u e n t i s introduced under very mild c o n d i t i o n s ( I C I , 0.5N HC1, 20°C) i n the t e r m i n a l step, t h i s sequence i s i d e a l l y s u i t e d f o r the s y n t h e s i s o f I7-MK-447. The a l t e r n a t i v e pathway, Xb >XIIb ( e i t h e r d i r e c t l y or stepwise v i a c h l o r o m e r c u r i a l X l l a ) >MK-447 a l ­ though q u i t e u s e f u l , i discussed below. The intermediat deactivated to e l e c t r o p h i l i c s u b s t i t u t i o n r e l a t i v e to phenol Xa and, t h e r e f o r e , X l l b r e q u i r e s more a c i d i c c o n d i t i o n s to f a c i l i t a t e the Tscherniac-Einhorn r e a c t i o n . The stronger a c i d medium i s conducive to d e i o d i n a t i o n and leads to reduced y i e l d s o f MK447. Nevertheless, the a l t e r n a t i v e pathway proved to be the route o f choice f o r e l a b o r a t i n g MK-447 l a b e l e d with a 2 - ^ £7-aminomethyl moiety. In t h i s i n s t a n c e , conversion o f o-iodophenol X l l b to Ζ^Ε?-ΜΚ-447 was achieved i n 35$ o v e r a l l y i e l d u s i n g N-(hydroxyC C^-methylàchloroacetamide which was conveniently generated i n s i t u from /Z _£7-paraformaldehyde and chloroacetamide. 1

MOLECULAR STRUCTURE, METABOLISM AND PHARMACOLOGY OF MK-447 Molecular S t r u c t u r e - As determined by potentiometric t i t r a t i o n i n water, MK-447 e x h i b i t s pKa-j^ 7.25 and p K a 10.75· These pKa values r e f l e c t the amphoteric p r o p e r t i e s o f MK-347 and, when compared with those (pKa, 8.4, 10.5) d i s p l a y e d by 2-aminomethylphenol hydrochloride, they i n d i c a t e that i n t r o d u c t i o n o f the 6-iodo s u b s t i t u e n t enhances the a c i d i t y o f the phenolic hydroxyl group or, viewed from a d i f f e r e n t p e r s p e c t i v e , serves to reduce net molecular b a s i c i t y , i . e . , monodeprotonation occurs a t a lower pH. When p a r t i t i o n e d between 1-octanol and pH 7.4 b u f f e r , MK-447 i s l o c a l i z e d e s s e n t i a l l y q u a n t i t a t i v e l y ( c a . 99$) i n the l i p i d phase. The f r e e base form o f MK-447, r e a d i l y l i b e r a t e d from MK-447 by n e u t r a l i z a t i o n with weak bases such as ammonia and sodium b i c a r bonate, i s very s o l u b l e i n non-polar organic s o l v e n t s and r e l a t i v e l y i n s o l u b l e i n aqueous media. The l i m i t e d water s o l u b i l i t y of MK-447 f r e e base suggests that i t has appreciable z w i t t e r i o n i c character i n water. Indeed, the v a l i d i t y o f t h i s suggestion was confirmed by determining the e f f e c t s o f pH on X f o r MK-447 i n water. The bathochromic s h i f t from 285 to 309 mywhich accompanies phenoxide formation ( i . e . , ArOH >Ar0~) was observed at pH 6. Hence, the observed s o l u b i l i t y c h a r a c t e r i s t i c s o f MK-447 f r e e base 2

2

m a Y

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7.

SMITH ET AL. 2-Aminomethylphenob

JH

NHC0CH C1

OH

2

EtOH-conc. HC1 (3:1),

: ( C H

107

ΝΗ ·Η01 2

Û C(CH )

3>3

3

Xlb

3

XIV

C1CH C0NHCH 0H, 2

2

HOAc, H S 0 2

4

ICI, 0.5 N HC1,

(cat.),

r.t r.t.

OH

H

NH *HC1 2

Ο CCCH ) 3

3

C(CH ) 3

Xb

2

MK-447 HgCOAc) ;

C1CH C0NHCH 0H,

ICI, HOAc,

2

NaCl, H 0

Δ

2

2

2

H0Ac-H S0 2

4

(10:1);

EtOH-conc. HC1 (3:1),

OH ClHgs

ο

I , 2

C(CH ) 3

HOAc

(CH ) 3

3

3

Xllb

Xlla

Figure 1.

Synthetic routes to MK-447

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

108

r e f l e c t i t s unique a b i l i t y t o a d j u s t molecular p o l a r i t y solvent-dependent manner as i l l u s t r a t e d i n equation 2. process should be q u i t e f a c i l e s i n c e , a p r i o r i , minimal would be r e q u i r e d t o e f f e c t the depicted i n t r a m o l e c u l a r transfer.

in a This energy proton

Examination o f th compound V l l i , c a l c u l a t e d by the CNDO/2 formalism (6) and pre­ sented as an ORTEP p r o j e c t i o n (7) i n F i g u r e 2, a f f o r d s a d d i t i o n a l i n s i g h t i n t o the physicochemical p r o p e r t i e s c i t e d above. First, the plane formed by the C1-C6-C1-0-H atom array i s coplanar with the aromatic r i n g and the p h e n o l i c hydrogen atom ( H ) i s hydrogen bonded t o the c h l o r o s u b s t i t u e n t . T h i s r e s u l t i s i n accord with the recent demonstration o f i n t r a m o l e c u l a r hydrogen bonding i n o-halophenols, i n c l u d i n g 2-iodophenols, by Kollman e t a l (8). Furthermore, although the aminomethyl group i s s i t u a t e d sub­ s t a n t i a l l y out o f the plane o f the r i n g (-9-= 4 5 ° ) , a second i n t r a m o l e c u l a r hydrogen bond e x i s t s between H^ o f the amino group and the p h e n o l i c oxygen atom. The s o l i d s t a t e s t r u c t u r e o f MK-447 f r e e base, determined by the s i n g l e c r y s t a l X-ray c r y s t a l l o g r a p h i c technique, i s i n reasonable agreement with the c a l c u l a t e d grounds t a t e s t r u c t u r e o f compound V l l i . A comparison o f these s t r u c ­ t u r e s , shown by t h e i r superimposition ( i . e . , one over the other) i n F i g u r e 3, i n d i c a t e s t h a t they d i f f e r i n two r e s p e c t s : (a) compound V l l i contains a H . . . C l , whereas, the -OH group i n MK-447 f r e e base i s not i n t r a m o l e c u l a r l y hyxirogen Bonded; i n s t e a d , i t i s d i r e c t e d below and perpendicular to the plane o f the a r o ­ matic r i n g and (b) the s i z e o f the d i h e d r a l angle -Θ-(45° v s . 53°) i s minimally d i f f e r e n t . The f i r s t s t r u c t u r a l d i f f e r e n c e i s somewhat s u r p r i s i n g and may w e l l r e f l e c t the inherent d i f f i c u l t i e s a s s o c i a t e d with a c c u r a t e l y l o c a t i n g a hydrogen atom proximate to an iodo s u b s t i t u e n t by X-ray c r y s t a l l o g r a p h y . The minor d i f ­ ference i n -Θ- could e i t h e r r e f l e c t the c r y s t a l packing f o r c e s i n the s o l i d s t a t e or stem from the well-known overestimation o f a t t r a c t i v e f o r c e s between non-bonded atoms by the CNDO/2 method (£). In any event, the s u b s t i t u t i o n o f iodo and aminomethyl groups v i c i n a l l y to the hydroxyl moiety f a c i l i t a t e s i n t r a m o l e c u l a r hydrogen bonding which, coupled with the presence o f a 4 - ( l , l dimethylethyl) s u b s t i t u e n t , imparts s u b s t a n t i a l l i p o p h i l i c i t y t o MK-447 f r e e base. Likewise, t h i s nuclear s u b s t i t u t i o n p a t t e r n i s &

x

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7. SMITH ET AL.

2-Amtnomethylphenols

Figure 2. The lowest energy ground state conformation of compound Vlli, the 6-chloro analog of MK-447 free base. This conformation, presented as an ORTEP projection, was calculated by the CNDO/2 semiempirical technique.

Figure 3. Comparison of the x-ray structure of MK447 free base (dotted lines) with the CNDO/2-calculated lowest energy ground state conformation of 6-chloro analog VIH (solid lines)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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110

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conducive to z w i t t e r i o n formation i n aqueous media and, thereby, s e v e r e l y l i m i t s the s o l u b i l i t y o f MK-447 f r e e base, but not that of the parent h y d r o c h l o r i d e , i n water. Metabolism - S t u d i e s Î10) i n r a t s , dogs and man i n d i c a t e that o r a l l y administered £ C7-MK-447 i s r a p i d l y absorbed, metabolized and excreted. Peak plasma drug l e v e l s were observed w i t h i n 1 t o 2 hr post drug a d m i n i s t r a t i o n i n a l l three species; a t . J h i s time, the parent drug accounted f o r c a . 15% o f the t o t a l C G7-radioa c t i v i t y i n human plasma and cajj,5$ o f that i n r a t and dog plasma. The h a l f - l i f e o f drug-related"~Zr C 7 r a d i o a c t i v i t y i n the plasma was about 7.5 hr i n man and dogs and 1 hr i n r a t s . In each o f these s p e c i e s , the p r i n c i p l e route o f drug e l i m i n a t i o n ( p r i m a r i l y as metabolites XV and XVI, v i d e i n f r a ) was v i a the u r i n e , whereas, the feces c o n s t i t u t e d the minor pathway f o r drug e x c r e t i o n As depicted i n Schem dogs almost e x c l u s i v e l (Compound XV). The major human metabolite o f MK-447 has been i s o l a t e d and t e n t a t i v e l y assigned s t r u c t u r e XVI, the N-glucuronide d e r i v a t i v e o f MK-447. The minor human metabolite corresponds to 0 - s u l f a t e e s t e r XV. The assignment o f s t r u c t u r e XV t o the major rat and dog metabolite was confirmed by d i r e c t comparison o f the i s o l a t e d metabolite with an authentic sample o f 2-aminomethyl-4-(l,l-dimethylethyl)-6-iodophenyl hydrogen s u l f a t e which was synthesized by the O-jsulf&tion process presented i n Scheme I I . I t should be noted that compound XV d i s p l a y s marked s a l i d i u r e t i c a c t i v i t y i n both r a t s ( s a l u r e t i c score = 3) and dogs ( s a l u r e t i c score = 5 ) . In view o f the data presented i n Table VII (vide supra) f o r O - s u b s t i t u t e d s a l i c y l a m i n e s Va-c, the observed a c t i v i t y a f t e r a d m i n i s t r a t i o n o f 0 - s u l f a t e e s t e r XV suggests that O - d e s u l f a t i o n occurs a t the proper s i t e i n v i v o , l i b e r a t i n g MK-447 i n agreement with the widely-accepted p r i n c i p l e o f microscopic r e v e r s i b i l i t y , a t l e a s t as i t a p p l i e s to enzymatic transformat i o n s . Hence, although u l t i m a t e l y e l i m i n a t e d v i a u r i n a r y and f e c a l e x c r e t i o n , metabolite XV may w e l l serve i n v i v o as both a depot and a pro-drug form o f MK-447. F i n a l l y , i n s p i t e o f i t s s t r u c t u r a l s i m i l a r i t y t o thyroxine, MK-447 does not undergo detectable d e i o d i n a t i o n e i t h e r i n v i t r o o r i n v i v o . Pharmacology (11) - O r a l l y administered i n both normotensive and spontaneously hypertensive r a t s , MK-447 d i s p l a y e d marked s a l u r e t i c and d i u r e t i c e f f e c t s which were-rapid i n onset and r e l a t i v e l y modest i n d u r a t i o n , the major a c t i o n having occurred w i t h i n the f i r s t 5 h r s . A comparison o f the s a l i d i u r e t i c a c t i v i t i e s o f MK-447, furosemide and h y d r o c h l o r o t h i a z i d e i n normotensive r a t s i s presented i n F i g u r e s 4, 5 and 6. F o r those r e n a l parameters measured, t h i s comparison demonstrates that (a) the c e i l i n g e f f e c t s o f MK-447 exceed those o f furosemide and hydrochlorot h i a z i d e and (b) MK-447 i s s i g n i f i c a n t l y more potent than f u r o semide. A p r e c i s e comparison o f the r e l a t i v e potencies o f MK-447

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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2-Aminomethylphenok

Scheme I I Metabolism of MK-447

Man

C(CH ) 3

3

MK-447

P y r i d i n e ^ S 0 , < 25°C 3

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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112

Na+ E x c r e t i o n mEq/cage/5 h r .

FUROSEMIDE

0.1

0.3

1.0

3.0

9.0

27.0

81.0

DOSE, mg/kg P.O. Figure 4. Dose-response regression lines for the natriuretic effects of orally-administered MK-447, furosemide, and hydrochlorothiazide in normotensive rats over a five-hr period. The data points are average values determined per cage for six to nine cages (three rats per cage of each drug. The placebo values for the same period were: Na\ 0.28; K\ 0.16; and Cl~, 0.25 mEq/cage, and urine volume, 28 mL/cage.

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SMITH ET AL. 2-Aminomethylphenols

7.

+

K Excretion mEq/cage/5 Hr.

0.1

0.3

1.0

3.0

9.0

27.0

81.0

DOSE, Mg/Kg P.O. Figure 5. Dose—response regression lines for the kaliuretic effects of orally-administered MK-447, furosemide, and hydrochlorothiazide in normotensive rats over a five-hr period. The data points are average values determined per cage for six to nine cages (three rats per cage) at each dose of each drug. The placebo values for the same period were: Ν a", 0.28; K , 0.16; and CI', 0.25 mEq/cage, and urine volume, 28 mL/cage. +

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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114

Figure 6. Dose-response regression lines for the chloruretic effects of orally-administered MK-447, furosemide, and hydrochlorothiazide in normotensive rats over a five-hr period. The data points are average values determined per cage for six to nine cages (three rats per cage) at each dose of each drug. The placebo values for the same period were: Na\ 0.28; K , 0.16; and CI; 0.25 mEq/cage, and urine volume, 28 mL/cage. +

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115

and h y d r o c h l o r o t h i a z i d e i s precluded by the l a c k o f slope p a r a l l e l i s m i n t h e i r dose-response r e g r e s s i o n l i n e s . Evidence that the s a l i d i u r e t i c a c t i v i t y o f MK-447 may r e l a t e t o i t s a b i l i t y t o enhance kidney l e v e l s o f PGE has been provided by Kuehl, e t a l . (12, 13) who showed that t h i s drug has the a b i l i t y t o enhance the s y n t h e s i s o f PGEs i n ram seminal v e s i c u l a r microsomes and i n i n c u b a t i n g kidney s l i c e s . A t y p i c a l experiment demonstrating the a b i l i t y o f MK-447 t o f a c i l i t a t e the conversion o f a r a c h i d o n i c a c i d t o PGE^ i s shown i n F i g u r e 8. The mechanistic d e t a i l s f o r t h i s a c t i o n o f MK-447 have been described (12, 14). Support f o r the concept that the s a l i d i u r e t i c a c t i o n o f MK-447 r e l a t e s t o PG p r o d u c t i o n i s provided by the f i n d i n g that treatment o f r a t s with indomethacin (2 mg/kg p.o.) 1 h r p r i o r t o dosing with MK-447 a f f e c t e d both e l e c t r o l y t e e x c r e t i o n and u r i n e volumes as shown on the s a l i d i u r e t i c a c t i o pretreatment with a lower dose (1 mg/kg p.o.) o f indomethacin d i d not s i g n i f i c a n t l y a f f e c t the potency o f MK-447. On the other hand, a t a dose o f 4 mg/kg p.o., indomethacin s u b s t a n t i a l l y reduced both the s a l u r e t i c and d i u r e t i c e f f e c t s o f MK-447. Furthermore, indomethacin (4 mg/kg p.o.) alone m a r g i n a l l y reduced r a t u r i n e volumes, which suggests that the observed e f f e c t o f indomethacin pretreatment may be due only p a r t i a l l y t o a s p e c i f i c antagonism o f the d i u r e t i c a c t i o n s o f MK-447. The s a l i d i u r e t i c e f f e c t s o f MK-447, furosemide and hydroc h l o r o t h i a z i d e g i v e n p.o. i n unanesthetized dogs a r e compared i n Table X I I I . At each o f the doses s t u d i e d , MK-447 d i s p l a y e d s a l u r e t i c and d i u r e t i c e f f e c t s g r e a t e r i n magnitude than those o f e i t h e r furosemide o r h y d r o c h l o r o t h i a z i d e . As observed i n r a t s , MK-447 e l i c i t e d s l i g h t l y more c h l o r u r e s i s than n a t r i u r e s i s i n dogs. K a l i u r e s i s was i n c r e a s e d s i g n i f i c a n t l y by each o f the three drugs. I n a n e s t h e t i z e d dogs, d o s e - r e l a t e d i n c r e a s e s i n e l e c t r o l y t e e x c r e t i o n and u r i n a r y volumes r e s u l t e d from MK-447 g i v e n i . v . over the e n t i r e 0.1 t o 25 mg/kg dose range. The Na /K e x c r e t i o n r a t i o approached 14 a t the h i g h e s t dose (25 mg/kg i . v . ) . When evaluated i n spontaneously h y p e r t e n s i v e (SH) r a t s , MK-447 (dose_> 0.312 mg/kg p.o.) e x h i b i t e d a n t i h y p e r t e n s i v e a c t i v i t y . At doses o f 1.25 and 5 mg/kg p.o., the a n t i h y p e r t e n s i v e e f f e c t s o f MK-447 were r a p i d i n onset ( w i t h i n 1 h r ) , pronounced i n potency and prolonged i n d u r a t i o n ( 24 h r ) . I n a d d i t i o n , the a n t i h y p e r t e n s i v e a c t i v i t y o f MK-447 i n SH r a t s was maintained upon repeated o r a l a d m i n i s t r a t i o n a t 0.312 mg/kg as demonstrated i n Table XIV. Under c o n d i t i o n s where MK-447 (0.312 mg/kg p.o.) produced a pronounced a n t i h y p e r t e n s i v e response, furosemide a t 20 mg/kg p.o. e x h i b i t e d no hypotensive effect. Evidence which suggests that the r e n a l p r o s t a g l a n d i n s , i n a d d i t i o n t o t h e i r p o s s i b l e c o n t r i b u t i o n t o the s a l i d i u r e t i c

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116

TABLE X I I Antagonism of the S a l u r e t i c and D i u r e t i c E f f e c t s of MK-447 by Indomethacin P r e t r e a t m e n t i n Normotensive Rats 3

b Values , 0-5 Hr P e r i o d

Excretion

Treatment

Dose (mg/kg p.o.) N a

+

(mEq)

K

+

(mEq)

Urine C l " (mEq) V o l (ml)

Placebo

0

MK-447

9

2.70

0.77

3.64

44

MK-447

27

3.19

0.88

4.35

47

MK-447

81

3.51

0.99

4.89

47

MK-447 + Indomethacin

9 2

2.52

0.85

3.59

42

MK-447 + Indomethacin

27 2

2.97

0.97

4.24

45

MK-447 + Indomethacin

81 2

3.25

1.02

4.70

43

Pretreatment 1 h r p r i o r to dosing with MK-447. per cage, 3 r a t s per cage.

Average values

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7. SMITH ET AL.

2-Aminomethylphenoh

117

TABLE X I I I S a l u r e t i c and D i u r e t i c E f f e c t s o f O r a l l y Administered MK-447 (MK), Furosemide (F) and H y d r o c h l o r o t h i a z i d e (HCT) i n Unanesthetized Dogs

a

E x c r e t i o n Values , 0-24 h r . P e r i o d Na

Dose (mg/kg) MK

0.2

10

0.3

16

+

(mEq) F

+

12

9

25

7

12 6

5

Urine V o l (ml)

17

260 12

8 11

22

(mEq)

HCT

15

1

CI

(mEq)

6

0.5 0.6

K

19

11

8

540 25

26

660

38

19

46

570

1.8

42

16

52

700

2

50

17

73

890

37

5

59

10

10 24

57

26

18

44 10

18

12

375 440

1.3

3

510

15 36

8

330

77

725 31

69

800

590

960

31

1035

760

Average values determined per dog f o r 3 to 47 dogs at each dose o f e a c h dru:^. The placebo values f o r the same p e r i o d were: Na , 4.8; Κ , 6.3; and C l " , 6.3 mEq/dog and u r i n e volume, 210 ml/dog. +

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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AGENTS

TABLE XIV A n t i h y p e r t e n s i v e A c t i v i t y of MK-447 Administered O r a l l y i n SH Rats of the Wistar-Okamoto S t r a i n

Group No. (No, o f Rats)

Treatment

Mean A r t e r i a l Pressure (mmHg + S E ) , Hr a f t e r Treatment

Day

a

0

1 (17)

Salin 2 ml/kg p.o.

MK-447

b

12

24

171+3

171+3

171+2

170+2

173+2

163+5

166+7

167+7

2

157+7

161+6

156+5

3

157+4

159+3

181+4

173+4

2

—

3 2 (5)

4

1

185+3

A r t e r i a l pressure was recorded i n unanesthetized male r a t s of 290 to 350 g body weight and 30 to 40 weeks age by a digect technique i n v o l v i n g c a n n u l a t i o n of the caudal a r t e r y . MK-447 (0.312 mg/kg) was administered p.o. i n water i n volumes of 2 ml/kg d a i l y f o r 3 days.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7.

SMITH ET AL.

I 0.01

:

119

2-Aminomethylphenoh

ι

1

0.1

10

10.0 00SE MG/KG P.O.

100.0

Figure 7. Dose-response regression lines for the effects of MK-447 on the mean arterial pressure and Na excretion in spontaneously hypertensive rats. The data points for decreases in mean arterial pressure are average values per rat for four to six rats per dose; the data points for Na+ excretion are average values per cage determined for nine cages (three rats per cage) at each dose. +

20

30 40 X I0" M ( Arochidonic Acid ) 5

Figure 8. The stimuhtory effects of MK447 on the biosynthesis of PGE in ram seminal vesicular microsomes as a func­ tion of substrate concentration

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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e f f e c t s o f MK-447 (vide s u p r a ) . may a l s o play a r o l e i n mediating the a n t i h y p e r t e n s i v e a c t i v i t y o f MK-447 emerged from s t u d i e s i n SH r a t s . In t h i s t e s t s p e c i e s , the marked a n t i h y p e r t e n s i v e e f f e c t s e l i c i t e d by 0.078, 0.312 and 1.25 mg/kg p.o. doses o f MK-447 were s e r i o u s l y attenuated by the o r a l c o a d m i n i s t r a t i o n of e i t h e r indomethacin (1.25 mg/kg) or a s p i r i n (20 mg/kg). I n t e r e s t i n g l y , MK-447 d i s p l a y e d no hypotensive a c t i v i t y i n normotensive Wistar-Kyoto r a t s a t doses up to 20 mg/kg p.o. Of even g r e a t e r pharmacological i n t e r e s t was the observation that MK-447 exhibi t e d a n t i h y p e r t e n s i v e a c t i v i t y i n SH r a t s a t s u b d i u r e t i c doses, i . e . , at doses t e n - f o l d lower than those r e q u i r e d f o r d i u r e s i s as shown by the dose-response r e g r e s s i o n l i n e s i n F i g u r e 7. The r e s u l t s c i t e d above i n d i c a t e t h a t , a t l e a s t i n SH r a t s , the a n t i h y p e r t e n s i v e a c t i v i t y o f MK-447 i s not s o l e l y dependent on i t s d i u r e t i c a c t i v i t y Further support f o r t h i s view emerges upon examination o f th a c t i v i t i e s recorded i d e r i v a t i v e s . Although compounds IXg, I X i , V l l i , V l l k and MK-447 d i s p l a y n e a r l y equipotent . s a l u r e t i c a c t i v i t i e s i n r a t s and dogs, t h e i r r e l a t i v e a n t i h y p e r t e n s i v e e f f e c t s a r e markedly d i f f e r e n t . Hence, as noted e a r l i e r , the pronounced a n t i h y p e r t e n s i v e propert i e s o f MK-447 served to d i s t i n g u i s h i t from a s u b s e r i e s o f potent s a l i d i u r e t i c agents which emerged from t h i s study. It is i n t e r e s t i n g to note t h a t the i n i t i a l s a l u r e t i c screening l e a d ( l a ) i s not a n t i h y p e r t e n s i v e i n the SH r a t , whereas, the parent s t r u c t u r e , s a l i c y l a m i n e (Compound V i a ) , has demonstrable,, a l b e i t weak, a n t i h y p e r t e n s i v e a c t i v i t y i n the SH r a t , but i t i s devoid of s a l u r e t i c a c t i v i t y . The t h i r d pharmacological a t t r i b u t e of MK-447, a n t i i n f l a m matory a c t i v i t y , was demonstrated by i t s e f f e c t i n reducing both carrageenan-induced f o o t edema i n r a t s and c r o t o n - o i l induced s w e l l i n g i n mouse ears (12, 13)· T h i s a c t i o n was suggested to a r i s e from the a b i l i t y o f MK-447 to scavenge an oxygen centered f r e e - r a d i c a l r e l e a s e d i n the conversion o f PGG^ to PGH^. Evidence has been provided to show that t h i s r a d i c a l i s an important inflammatory mediator (12, 13). The i n i t i a l c l i n i c a l study (15) i n normal v o l u n t e e r s has shown t h a t MK-447 i s a potent, h i g h - c e i l i n g d i u r e t i c i n man as i n d i c a t e d by the n a t r i u r e t i c and k a l i u r e t i c data presented i n F i g u r e 9. In t h i s study, MK-447 d i s p l a y e d marked d o s e - r e l a t e d s a l i u r e s i s and d i u r e s i s with minimal k a l i u r e s i s . Recently, MK-447 was shown to e l i c i t a n t i h y p e r t e n s i v e a c t i v i t y a t d i u r e t i c doses i n man (16). MK-447 i s p r e s e n t l y undergoing f u r t h e r c l i n i c a l i n v e s t i g a t i o n . Whether the a d j u n c t i v e antiinflammatory a c t i v i t y demonstrated f o r MK-447 i n experimental animals w i l l . b e observed i n man i s yet to be e s t a b l i s h e d . Acknowledgments - We a r e indebted to Ms. S. J . deSolms, Mr. A. A. Deana and Mr. N. P. Gould f o r expert s y n t h e t i c a s s i s t a n c e , t o Mr. E. L. Cresson f o r the UV s t u d i e s , to Dr. L. S. Watson (deceased) and to Mr. H. F. Russo f o r the s a l i d i u r e t i c

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

7. SMITH ET AL.

TABLE XV. Compd.

a

2-Aminomethylphenoh

121

R e l a t i v e S a l u r e t i c and A n t i h y p e r t e n s i v e

Structure

S a l u r e t i c Score Rat Dog

Activity

Antihypertensive Score SH Rat RH Dog a

R e l a t i v e s c o r e s : outstanding a c t i v i t y = 3; moderate a c t i v i t y = 2; weak a c t i v i t y = 1; i n a c t i v e = 0.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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122

Δ Excretion (Drug-Control), mEq/4 Hr.

240h

200h

6.25

12.5

25.0

50.0

100

DOSE, mg, P.O. Figure 9. The natriuretic and kaliuretic effects of orally-administered MK-447 in nor­ mal human volunteers over a four-hr period. The data points are average values ( Δ excretion = drug control) determined per volunteer in eight volunteers at each dose. Derived from the data of Affrime, M. B., et al. (15).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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123

e v a l u a t i o n s , to Dr. A. S c r i a b i n e and to Mr. C . T. Ludden f o r the a n t i h y p e r t e n s i v e s t u d i e s and t o Dr. C. G. Van Arman f o r the antiinflammatory r e s u l t s . G r a t i t u d e i s expressed to Dr. J. L. Humes f o r p r o v i d i n g F i g u r e 8, t o Dr. K. Hoogsteen, Dr. J. Springer and Mr. J. H i r s c h f i e l d f o r the X-ray c r y s t a l l o g r a p h i c a n a l y s i s o f MK-447 f r e e base and t o Dr. G. M. Smith f o r the CNDO/2 modeling s t u d i e s . We wish t o express our a p p r e c i a t i o n to Drs. D. J. Tocco and J. E. Baer and t h e i r colleagues f o r the metabolism s t u d i e s , to Dr. R. 0. Davies, Dr. J. M. Schrogie, Dr. Κ. E. Tempero and Dr. B. L e i f o r the c l i n i c a l i n v e s t i g a t i o n s and to Dr. J. M. Sprague ( r e t i r e d ) , Dr. C. A. Stone and Dr. R. F. Hirschmann f o r t h e i r guidance and encouragement throughout the course o f t h i s i n v e s t i g a t i o n .

Literature Cited and Notes 1. A series of manuscript describin th detail f thi investigation is in preparatio Ε. Μ., Deana, Α. Α . , deSolms, J., Sprague, , , R. L . and Cragoe, E. J., Jr., for submission to J. Med. Chem. 2. Meadow, J., Berger, J. and Schert, R., Chim. Therap. (1968), 3, 253. 3. Geschickter, C. and Meadow, J., U.S. Patent 3,080,365 (1968). 4. For an extensive review of the Tscherniac-Einhorn reaction, see Zaugg, H. E. and Martin, W. B. in "Organic Reactions", 14, Adams, R., Blatt, A. H . , Boekelheide, V . , Cairns, T. L., Cope, A. C. and Niemann, C., Eds., J. Wiley and Sons, New York, Ν. Y . , 1965, pp 52-269. 5. Cragoe, E. J., J r . and Schultz, Ε. Μ., U.S. Patent 4,029,816 (1977). 6. Since the Merck Molecular Modeling CNDO/2 program was not parameterized for third and fourth row elements at the time of this study, 6-chloro analog VIIi was subjected to conformational analysis by the CNDO/2 calculational technique; Smith, G. W., unpublished results. 7. Johnson, C. K . , ORTEP, Rep. ORNL-3894 (1965), Oak Ridge National Laboratory, Oak Ridge, Tenn. 8. Dietrich, S. W., Jorgensen, S. W., Kollman, P. A. and Rothenberg, S., J. Am. Chem. Soc. (1976), 98, 8310. 9. Gregory, A. R. and Paddon-Row, M. W., J. Am. Chem. Soc. (1976), 98, 7521. 10. Tocco, D. J., Walker, R. W., Arison, Β. Η., VandenHeuvel, W. J. Α . , Stokker, G. E. and Smith, R. L., for submission to Drug Dispos. Metab. 11. Scriabine, Α . , Watson, L . S., Russo, H. F., Ludden, C. T . , Sweet, C. S., F a n e l l i , G. M . , Jr., Bohidar, N. and Stone, C. Α . , Fed. Proc. (1978), 37, 921. 12. Kuehl, F . Α . , Jr., Humes, J. L., Egan, R. W., Ham, E. Α . , Beveridge, G. C. and Van Arman, C. G . , Nature (1977), 265, 170. 13. Kuehl, F . Α . , Jr., Egan, R. W., Humes, J. L., Beveridge,

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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G. C. and Van Arman, C. G . , in "New Biochemical Aspects of Prostaglandins and Thromboxanes", Fried, J. and Kharasch, N . , Eds., Academic Press, New York, Ν. Y . , 1978. 14. Kuehl, F . Α . , Jr., Oien, H. G. and Ham, E. A. in "Prosta­ glandins in Cardiovascular and Renal Function", A. Scriabine, F. A. Kuehl, J r . and A. M. Lefer, Eds., Spectrum Publications, New York, 1978, in press. 15. Affrime, M. B., Lowenthal, D. T., Onesti, G . , Busby, P . , Schwartz, C. and L e i , B., C l i n . Pharmacol. Ther. (1977), 21, 97. 16. Davies, R. O., personal communication (to be published). RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

8 1-Aralkyl-2-pyrazolin-5-ones: A New Class of Highly Potent Diuretics with High Ceiling Activity H. HORSTMANN, E . MÖLLER, E . WEHINGER, and K. M E N G Bayer Pharma Research Center, Postfach 10 17 09, D-56 Wuppertal 1, Federal Republic of Germany

The diuretics in pharmacologically and c l i n i c a l l -ceiling or high-ceiling a c t i v i t y . Compounds having a low-ceiling activity exhibit a leveling-off or plateau in their diuretic action. Beyond a certain point, the diuretic effect i s not increased by an increase in dosage. Typical low ceiling diuretics are the thiazides. Also, such compounds as chlorthalidone and mefruside belong to this group. A further characteristic of this group is the protracted duration of activity which i s particularly advantageous in long-term treatment (Figure 1). In contrast to this, the dose/activity curves of high-ceiling diuretics run almost linearly over a wide range. Such diuretics have a reserve in capacity and can, therefore, also be used on thiazide-resistant patients. A major disadvantage i s their short duration of activity which i s coupled with rebound phenomena. Representative compounds having this type of activity are ethacrynic acid, furosemide and bumetanide. The medically most desirable combination of high-ceiling activity and protracted duration could not be realized until now. This was, therefore, the target of our own work in this area. While it seemed improbable that progress could be made in this direction merely by working on known diuretics, we concentrated our attention on new classes of active substances. Structural elements such as 1 and 2 which appear frequently in diuretic purines, triazines, pteridines, pyrazines and

>N-C=N-

C. H N' y

,

2

1

2

pyrimidines, served as g u i d e l i n e s f o r s e l e c t i o n . 0-8412-0464-0/78/47-083-125$05.00/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

126

A number o f new d i u r e t i c s , such as furosemide, bumetanide and t r i f l o c i n (JL), have amphoteric p r o p e r t i e s , so we a l s o looked c l o s e l y at compounds having a c i d i c and b a s i c f u n c t i o n s i n the same molecule. As a r e s u l t o f t h i s extensive search, we discovered a new c l a s s o f agents represented by compound 3 a few years ago which possessed moderate d i u r e t i c a c t i v i t y i n r a t s and dogs.

3 As may be seen, t h i s i tuted i n the 1 - p o s i t i o In c o n t r a s t to the well-known and thoroughly i n v e s t i g a t e d 3-amino-l-arylpyrazolin-5-ones, 3-amino-l-aralkylpyrazolin-5-ones had only been described i n a few i n s t a n c e s i n the patent l i t e r ature at the s t a r t o f our work, and then, only as coupling components f o r magenta d y e s t u f f s ( 2 ) . The chemistry o f t h i s c l a s s w i l l t h e r e f o r e be discussed f i r s t . The p r e p a r a t i o n of 3-aminopyrazolin-5-ones (Formula 5) bearing aromatic s u b s t i t u e n t s i n the 1 - p o s i t i o n has been s t u d i e d e x t e n s i v e l y by Weissberger and co-workers (3, 5) and i s c a r r i e d out by r e a c t i o n of a r y l h y d r a z i n e s with cyanoacetate (Compound 4) i n the presence o f a l k o x i d e . C e r t a i n h e t e r o c y c l i c hydrazine d e r i v a t i v e s , such as 2 - p y r i d y l h y d r a z i n e , y i e l d the isomeric 3-hydroxypyrazolin-5-imines (Formula 6) under these c o n d i t i o n s .

6

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

8. HORSTMANN ET AL.

l-Aralkyl-2-pyrazolin-5-ones

127

I f 3-amino-3-ethoxyacrylate (Compound 7) i s employed i n s t e a d o f compound 4, then the 3-aminopyrazolin-5-one analogous to compound 5 i s formed i n both cases. In c o n t r a s t to t h i s behavior, a l i p h a t i c hydrazines, such as methylhydrazine (Compound 8 ) , r e a c t even with compound 7 to give a mixture o f 3-aminopyrazolin-5-one (Compound 9) and 3-hydroxypyrazolin-5-imine (Compound 10).

7

8

9

10

T h i s r e s u l t shows that bot r e a c t i n g e i t h e r with th t i o n o f compound 7. One would expect a s i m i l a r behavior from aralkylhydrazines. I f one r e a c t s 4-chlorobenzylhydrazine (Compound 11) with compound 7 under m i l d c o n d i t i o n s f o r a short time and t r a p s the primary product (Compound 12) with 4 - n i t r o b e n z a l d e hyde, then one i s o l a t e s the hydrazone (Compound 13) i n good y i e l d .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

128

AGENTS

Under f o r c i n g c o n d i t i o n s , compound 7 and compound 11 r e a c t smoothly t o give compound 3 i n good y i e l d . T h i s shows t h a t the e s t e r f u n c t i o n r e a c t s p r i m a r i l y and that the favored point o f attack i s the s u b s t i t u t e d N-atom of compound 11. I f the imidate f u n c t i o n i s a c t i v a t e d by s a l t formation, then the amidrazone (Compound 14) i s formed p r i m a r i l y , which does not r e a c t with aldehydes. I n t h i s case, the unsubstituted N-atom of compound 11 i s the favored p o i n t o f a t t a c k .

7 -HC1 + 11

f^NH-NH-CH -/o\-Cl 2

0

^OCOHR

•HC1

Ik

OH"

5 On a d d i t i o n o f a l k o x i d e , compound 14 i s c y c l i z e d spontaneously to compound 3 so that the same product r e s u l t s from both r e a c t i o n s . I f a r a l k y l h y d r a z i n e s are reacted d i r e c t l y with compound 4 i n the presence o f a l k o x i d e , then mixtures o f the isomeric pyrazol i n e s (Compounds 16 and 17) are formed, as shown i n the r e a c t i o n of 3,4-dichlorobenzylhydrazine (Compound 15). NH

2

4

15

16

17

Since compound 3 i s a p o l y f u n c t i o n a l s p e c i e s , i t o f f e r s countless o p p o r t u n i t i e s f o r molecular v a r i a t i o n ; t h e r e f o r e , a r e p r e s e n t a t i v e s e l e c t i o n o f analogs w i l l be made f o r d i s c u s s i n g the e f f e c t o f s t r u c t u r a l changes on d i u r e t i c a c t i v i t y . The general formula 18 serves as a b a s i s f o r t h i s d i s c u s s i o n .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

8.

HORSTMANN ET AL.

l-Aralkyl-2-pyrazolin-5'Ones

In Table I , the q u a l i t a t i v e r e l a t i o n s h i p i s presented between s t r u c t u r e and a c t i v i t y f o r the p y r a z o l i n - 5 - o n e s ( S t r u c t u r e 19) a f u n c t i o n of the s u b s t i t u t i o i th 3 - p o s i t i o n

129

as

The r e s u l t s show that a l k y l a t i o n or a c y l a t i o n of the amino group causes a d r a s t i c l o s s of a c t i v i t y . Replacement of amino by a hydroxy- or carbonyl group or groups d e r i v e d from these m o i e t i e s a l s o leads to weak or i n a c t i v e compounds. In t h i s regard, i t was a l l the more s u r p r i s i n g that compound 26 (and s i m i l a r p y r a z o l i n 5-ones s u b s t i t u t e d by lower a l k y l groups) possessed a noteworthy d i u r e t i c potency. A p l a u s i b l e e x p l a n a t i o n f o r these r e s u l t s i s not p o s s i b l e at the present time. The carbonyl group i n the 5 - p o s i t i o n i s of c r u c i a l importance f o r a c t i v i t y . I n t e r a l i a , t h i s may be seen by the f a c t that l - ( 4 - c h l o r o b e n z y l ) - 3 - h y d r o x y p y r a z o l i n - 5 - i m i n e , i s o m e r i c with Compound 3, and the corresponding 3 , 4 - d i c h l o r o b e n z y l d e r i v a t i v e (Compound 15) have no d i u r e t i c a c t i v i t y . Furthermore, 3-aminol - ( 4 - c h l o r o b e n z y l ) p y r a z o l i n - 5 - i m i n e , prepared from 4-chlorobenzylhydrazine and m a l o n i t r i l e , i s a l s o i n a c t i v e . 3-Aminopyrazolin-5-ones ( S t r u c t u r e 32) possess amphoteric p r o p e r t i e s . I t has been shown by NMR-studies that the e q u i l i b rium Compound 32 ^ z z ^ Compound 33 l i e s i n f a v o r of the enol form (Compound 33) i n p o l a r s o l v e n t s .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

130

DIURETIC

AGENTS

TABLE I The Q u a l i t a t i v e R e l a t i o n s h i p between S t r u c t u r e and A c t i v i t y f o r the Pyrazolin-5-ones (Structure 19) with V a r i a t i o n s (R) i n the 3 - p o s i t i o n .

No.

R

3

-NH

20

-NHCH

+

2

23

3 -NHC H *5 -NH-COCH. 3 -NH-C0-NHCH

24

-OH

25

-0C H

26

-CH

21 22

2 7

6

5

3

o

C

c

3

H

" 6 5

28

-COOC H

29

-C00H

30

-C0NH

31

-C0NHC-NH II NH

2

5

2

o

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1 ~Aralkyl-2-pyrazolin-5-ones

HORSTMANN ET AL.

Effect

f

high ceiling activity S /

/

Furosemide Etacrynic A c i d

/

low ceiling activity

Dose Figure 1.

Dose-effect curves of diuretics with high-ceiling and low-ceiling activity

H Θ r (

4-ÇI —

~

+7Γ-αΛ

V branch

ι I —

4-OCHo

7

~

C

ι

© ~

—

+π-αΛ

V branch

7

ι' 1

~

π

Λ +7Π+σΛ V branch

4-CHo

1 I —

ιI

3 4-CI f

!

2

4-CF 3-CF ,4-CI Topliss-operation scheme greatly simpli­ fied 3

Figure 2.

7

3

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

132

AGENTS

-NHg

R

52

33

Our current data i n d i c a t e that the c a p a b i l i t y o f the com­ pounds to undergo e n o l i z a t i o n seems to be o f c r i t i c a l importance for a c t i v i t y . Inter a l i a thi als b concluded fro th f a c t that 3-aminopyrazolin have d i u r e t i c a c t i v i t y , , analog dialkylate 4 - p o s i t i o n are i n a c t i v e . Replacement o f the e n o l i c hydroxyl group by a c h l o r i n e atom a l s o leads to l o s s of a c t i v i t y . The s u b s t i t u t i o n p a t t e r n i n the aromatic residue has been e x t e n s i v e l y i n v e s t i g a t e d . In these s t u d i e s , the operation scheme developed by T o p l i s s (6) y i e l d e d good r e s u l t s . By the a p p l i c a t i o n of a somewhat s i m p l i f i e d " T o p l i s s t r e e (Figure 2) to d i f f e r e n t subgroups of pyrazoles, we found t h a t one always approached the area o f optimal a c t i v i t y by f o l l o w i n g the +π, +6-branch as f a r as the 3 , 4 - d i c h l o r o d e r i v a t i v e . Figure 3 shows the connection between the n a t u r a l logarithm of the sodium e x c r e t i o n i n the dog a f t e r o r a l a d m i n i s t r a t i o n o f 3 mg/kg of each substance and the l i p o p h i l i c i t y i n the s e r i e s of 3-amino-l-benzylpyrazolin-5-ones. The R value serves here as a measure o f the l i p o p h i l i c i t y (Figure 3). I f one takes i n t o account that these data were o r i e n t a t i n g values obtained on the i n t a c t animal, i . e . , two dogs per substance, then the r e s u l t , with a c o r r e l a t i o n c o e f f i c i e n t of 0.63 and a random sample s i z e o f 24, may be regarded as s a t i s f a c t o r y and serve as an a i d f o r planning new syntheses. The r e s u l t s would be b e t t e r i f the pharmacological data could be r e f i n e d by m u l t i p l e r e p e t i t i o n . T h i s was only p o s s i b l e with a small number of compounds due to l i m i t e d c a p a c i t y . We obtained very i n t e r e s t i n g r e s u l t s by v a r i a t i o n of the bridge X between the h e t e r o c y c l i c and a r y l m o i e t i e s . A s e l e c t i o n of compounds of general formula 34, which serves to i l l u s t r a t e t h i s , i s contained i n Table I I . 11

M

3h

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

8. HORSTMANN ET AL.

l-Aralkyl-2-pyrazolin-5-ones

133

TABLE I I The Average A c t i v i t i e s o f Compounds on V a r i a t i o n of the Bridge X between Heterocycle and A r y l Residue (Formula 34).

Subgroup

-

-

I II

-CH -

+

2

III

-CH(CH )-

+++

IV

-CH -CH -

+

V

-CH -CH -0-

++

VI

-CH -CH=CH-

++

VII

-CH -CH -CH -

-

3

2

2

2

2

2

2

2

2

*dog, 3 mg/kg p.o.; -

<500 yEq

+

Na /Kg

+

500-1000

++

1000-2000

»

>2000

»

+++

»

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

134

AGENTS

In Na+-Excretion (Dog) 3 mg/kg p.o. 8,0

, ,

7,0

03-Br,4-CI ...,..··"" 3-CI, 4-CI · · · 3-Br, 4-Br " 3-01· 3-CI,4-Br ,,...··*·*" 3,4-(CH = CH) * 3.4-(CH ) 4-Br# • 3-ci, 5-ci In N a = 3,7 R + 5,6 ,3 * ·3,4-(ΟΗ ) r =0,63 3-CH„4 "CH 4-CH(CH3) η =24 >3-CF, ...«"·' 4-CI NHo 2

2

3

+

M

4

P

3

2

• 4-OCH. 3 5,0

• 2-J • 4-0CF-

• 4-CH, 3-CH.

"VF* 4,0 H

3.0

2

#

6,0 H

0<Sl'

N

• »3.4-0CH-0Ri 1 -0,2

Figure 3.

1 -0,1

1 0,0

1 0,1

1 0,2

1 0,3

1 0,4

R

2

• R -Value (dioxane/H 0) M

2

Quantitative structure activity relationship between sodium excretion in the dog and lipophilicity

Figure 4.

X-ray crystal structure of muzolimine

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

8.

HORSTMANN ET AL.

l-Aralkyl-2-pyrazolin-5-ones

135

The r e s u l t s should, however, only i n d i c a t e a trend i n a c t i v i t y which was observed i n the dog by v a r i a t i o n of the bridge X, (Within the i n d i v i d u a l subgroups, the average was taken o f the r e s u l t s obtained on compounds with d i f f e r e n t s u b s t i t u t i o n pat­ terns . ) An important g u i d e l i n e f o r continued work i n t h i s c l a s s of compounds was the o b s e r v a t i o n that 3-amino-l-arylpyrazolin-5-ones (Subgroup I) were i n a c t i v e ; 3-amino-l-benzylpyrazolin-5-ones with a methyl branching i n the methylene group (Subgroup I I I ) were highly active. T h i s i s i l l u s t r a t e d by the comparison of some unbranched 3-amino-l-benzylpyrazolin-5-ones with the corresponding branched d e r i v a t i v e s (Table I I I , General Formula 35).

ι X-Ar 35 From the s e v e r a l hundred pyrazolin-5-ones s y n t h e s i z e d i n these l a b o r a t o r i e s , muzolimine (BAY g 2821, Compound 38, 3-aminol-(3,4-dichloro-a-methylbenzyl)pyrazolin-5-one) was s e l e c t e d f o r clinical trials.

38 F i g u r e 4 shows the X-ray c r y s t a l s t r u c t u r e of muzolimine. In Table IV, some physicochemical data f o r muzolimine and furosemide are listed. These data show that BAY g 2821 i s n o t i c e a b l y more l i p o p h i l i c than furosemide, which i s probably o f g r e a t s i g n i f ­ icance i n e x p l a i n i n g t h e i r d i f f e r e n t pharmacokinetics. In F i g u r e 5, the d o s e / a c t i v i t y curve i n the dog i s shown f o r different electrolytes. As can be seen, doses of muzolimine i n the range 0.1 mg/kg to 3 mg/kg cause an almost l i n e a r i n c r e a s e i n the e x c r e t i o n r a t e of c h l o r i d e and sodium i o n s . The potassium e x c r e t i o n i s only s i g n i f i c a n t l y i n c r e a s e d by doses above 1 mg/kg. The bicarbonate e x c r e t i o n remains almost u n a f f e c t e d . Thus, muzol­ imine behaves s i m i l a r to furosemide and must be added to the group of "high c e i l i n g " d i u r e t i c s . An even c l e a r e r demarcation with r e s p e c t to furosemide and the s p e c i a l a c t i v i t y p r o f i l e of BAY g 2821 i s seen when one s t u d i e s the a c t i v i t y vs. time curve a f t e r a s i n g l e dose o f 1 mg/kg. When one measures the e x c r e t i o n volume and e l e c t r o l y t e

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

TABLE I I I Comparison o f the A c t i v i t i e s o f Some Unbranched 3-Amino-l-Benzylpyrazolin-5-0nes with the A c t i v i t i e s of the Corresponding Branched D e r i v a t i v e s (Formula 35).

No.

-X-

-Ar-

3

-CH -

36

-CH(CH )-

37

-CH -

Activity*

2

+++

3

-<§>-

2

cl

++++

^Cl

38

-CH(CH )-

++++++ (muzolimine)

39

-CH -

+

HO

-CH(CH )-

3

2

O0r

3

++++

•Increased sodium e x c r e t i o n ; dog, 3 mg/kg p . o . / l nr. +

+ = 500 + 100 yEq Na /Kg

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

HORSTMANN ET AL.

l-Araïkyl-2-pyrazolin-S-ones

TABLE IV Physico-chemical P r o p e r t i e s o f Muzolimine i n Comparison with Furosemide

l g Ρ (octanol/H 0) 2

Furosemide

Muzolimine

3.9

9.2

-83

2.29

-.67

-.33

R.. (ethanol/H 0) o

R

M

(dioxane/H 0) 2

ElectrolyteExcretion m Eq/kg/3 hr

Co. 0,1 Figure 5.

0.3

1,0

3,0 mg/kg p.o.

Dose-activity curves in the dog after appli­ cation of muzolimine

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

138

AGENTS

Na*-Excretion mEq/kg/15 min. 1,4η

o

;

Figure 6. Time-response curves in the dog without substitution after a single dose of 1 mg/kg

ι—ι—ι—ι

-30

0

•-•BAYg2821,1 mg/kg i.v. 0--0Furosemide. 1 mg/kg i.v.

ι

30

ι

ι—ι—ι—ι—ι—ι—ι—ι—I

60

90 120 150 180 min. post inj.

Ν a -Excretion mEq/kg/15 min. ο 1.8i +

•BAYg2821,1 mg/kg i.v. •°Furosemide, 1 mg/kg i. v.

1,6 1,4

1,24 1.0H 0,8 0,6

0A-\ 0,2 Η 0 Figure 7. Time-response curves in the -ι—ι—ι—I 30 0 30 dog with substitution after a single dose of 1 mg/kg J

60

90

120 150 180 min. post inj.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

8.

HORSTMANN ET AL.

l-Aralkyl-2-pyrazolin-5-ones

139

q u a n t i t i e s at short i n t e r v a l s over 180 minutes, i t i s found that the a c t i o n o f furosemide i s o f r a p i d onset but soon drops away, whereas, with muzolimine, the i n i t i a l increase i n a c t i o n i s slower, but the d u r a t i o n i s longer (Figure 6 ) . These d i f f e r e n c e s become even more d i s t i n c t when the s a l t and volume l o s s e s caused by the substance are r e p l a c e d by an i n f u s i o n c a r e f u l l y matched t o the e x c r e t i o n r a t e . By t h i s means, the c o u n t e r - r e g u l a t i o n e f f e c t i n the healthy animal i s n u l l i f i e d and the s a l t and water reserves present i n the edematous p a t i e n t are simulated. Under these c o n d i t i o n s , one can see that the a c t i v i t y o f BAY g 2821 i s not only o f d i s t i n c t l y longer d u r a t i o n , but i s a l s o higher than that o f furosemide with r e s p e c t to the t o t a l e x c r e t i o n (Figure 7 ) . For approximately two years, muzolimine has been under c l i n i c a l t e s t . A whole s e r i e f carefull controlled studie have been completed i n (7-l^). In these s t u d i e s , y , especially the novel combination o f h i g h - c e i l i n g and l o n g - a c t i n g a c t i v i t i e s on d i f f e r e n t forms o f edema, p a r t i c u l a r l y on c a r d i a c and hepato­ genic edemas, has been confirmed.

Literature Cited 1. Meng, K. and Loew, D . , "Diuretika: Chemie, Pharmakologie, Therapie," 232, Georg Thieme Verlag, Stuttgart (1974). 2. Vanden Eynde, Η. Α . , Pollet, R. J. and DeCat, A. H., U.S. Patent 3,563,745 (1971). 3. Weissberger, A. and Porter, H. D . , J. Amer. Chem. Soc. (1942), 64, 2133. 4. Weissberger, Α . , Porter, H. D. and Gregory, W. Α . , J. Amer. Chem. Soc. (1944), 66, 1851. 5. Graham, B . , Porter H. D. and Weissberger, Α . , J. Amer. Chem. Soc. (1949), 71, 983. 6. Topliss, J. G . , J. Med. Chem. (1972), 15, 1006. 7. M ö l l e r , E., Horstmann, H., Meng, K. and Loew, D . , Experientia (1977), 33, 382. 8. Berg, K. J., Jorstad, S. and Tromsdal, Α . , Pharmatherapeutica (1976), 1, 319. 9. Fauchald, P. and Lind, E., Pharmatherapeutica (1977), 1, 409. 10. Hoppeseyler, G . , Heissler, Α . , Coppencastrop, Μ., Schindler, M. Schollmeyer, P. and Ritter, W., Pharmatherapeutica (1977), 1, 422. 11. Loew, D . , Curr. Med. Res. Opin. (1977), 4, 455. 12. Loew, D. and Meng, Κ., Pharmatherapeutica (1977), 1, 333. 13. Loew, D. and Meng, K . , Naunyn-Schmiedeberg's Arch. Pharmacol. (1977), 297, (Suppl. 2), R38. RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9 Quincarbate: A Representative of a New Class of Diuretics with 1,4-Dioxino[2,3-g]quinolone Structure 1

TH. A. C. BOSCHMAN, J. VAN DIJK , J. HARTOG, and J. N. WALOP Philips-Duphar B.V., Research Laboratories, 1380 AA, Weesp, The Netherlands

During an investigation of new quinolone derivatives (Figure 1), which were synthesized mainly for anticoccidiosis testing, i t was found that some of activity (1). In this paper, we w i l l discuss, in turn: (a) the structure-activity relationships (SAR) of the compounds of the series, (b) the physicochemical properties of some representative compounds, (c) the pharmacological profile and the metabolism of quincarbate, the compound selected for human pharmacological investigation and (d) the preliminary results of the evaluation of quincarbate i n humans. STRUCTURE-ACTIVITY RELATIONSHIPS The diuretic activity was determined in saline loaded rats after oral administration of the compounds according to a modification of the method of Lipschitz (2). The volume of excreted urine as well as i t s electrolyte contents were measured over a 5-hour period. The activity was expressed as an ED value, i.e., the dose (mg/kg) which gave a 100% increase i n the urinary volume over the control value. Compound 1 (Figure 2) i s a prototype structure which possesses the characteristics essential for diuretic a c t i v i t y . Variation of the ring structure, as well as, of the location of the substituents generally led to compounds with no oral activity at 50 mg/kg (Figure 3). Reorientation of the EtOCH -group to the v i c i n a l carbon atom in the dioxino ring, as in compound 2, or the reorientation of the carboxyl group in the pyridone ring, as in compound 3, produced a considerable decrease in activity or to inactive compounds. Contraction (Compound 4), expansion (Compound 5), or opening (Compound 6), or reorientation (Compound 7) of the dioxino ring also produced inactive compounds. Likewise, the presence of methyl groups on the pyridone nucleus had a disastrous effect on the a c t i v i t y (Compounds 8 and 9). Variation of R revealed that a small alkoxyalkyl substituent 200

2

3

1

Address correspondence to this author. 0-8412-0464-0/78/47-083-140$05.00/0 © American Chemical Society

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9.

BOSCHMAN ET AL.

Quincarbate

141

Figure 3. Inactive (a, b) analogs in which substituents or rings have been rearranged. (a) Diurèses estimated in saline-loaded rats; (b) inactive means: no activity at 50 mg/kg, orally.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

142

was e s s e n t i a l , since compounds 15, 16, 17, 18 and 19 were i n ­ a c t i v e , and i t a l s o showed that the oxymethylene bridge appeared optimal (Table I ) . Table I .

E f f e c t o f v a r i a t i o n o f RQ i n 0 lOOEt

no. * 3

1 10 11

ED o

no. R3

3.8 10

15 16

15

18 19

2 0

EtO-CH

2

H0-CH 2

ED200 (mg/kg)

Ί

H

>50

Me0-CH 2

compounds

12 13

MeO-CHg-CH -0-CH -

Ik

Et0-CH -CH -

PrO-CHg2

2

2

h

MeO-COn-CsHiT-O-CHg- >

20

2

The s u b s t i t u e n t R Q (Table I I ) seemed to be l e s s c r i t i c a l , since the carboxy or ethoxy carbonyl f u n c t i o n can be r e p l a c e d by an a c e t y l group (Compound 21) o r even by an a l k y l group (Compound 24). Whether the a c t i v i t i e s o f these v a r i o u s compounds a r e due t o metabolism has not been e s t a b l i s h e d . Table I I .

E f f e c t o f v a r i a t i o n o f Re i n 0 Re

no. Re

ED oo 2

1 20 21 22 23

-CHOH-Me

2k

-Et

-COOEt -C00H -CO-Me -CH -0H 2

3.8 7 12 4 11 10

ED200 (mg/kg)

no. Re

25 26 27 28 29 30 31

-C0-NH

2

-CsN -CHg-NHg

>50 or • compounds inactive

-H -Br -NHg -N0

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9.

BOSCHMAN ET AL.

143

Quincarbate

S u b s t i t u t i o n o f the benzene nucleus produced (Table I I I ) i n d i u r e t i c a c t i v i t y . Table I I I .

E f f e c t o f v a r i a t i o n o f R5 and R

1 0

marked change

in

COOEt Et0-CH

no.

R-io(Rs^L)

1 52 33 34 35 36 37

H He CI Br CF N0 NHAc

2

ED o

no.

Rio

R5

ED

3.8

38

NH

H

50

kl

Cl

20

2

2 0 0

(mg/kg)

k

0.08

rvO.k rJ 0.5

3

Cl

inactive compounds

^3 13

2

When R was a l i p o p h i l i c , e l e c t r o n e g a t i v e group, as i n compounds 33, 34, 35 and 36, a c t i v i t y was i n c r e a s e d , e s p e c i a l l y when R =C1, as i n compound 33. Compound 33 (whose recommended INN name i s quincarbate) was the most a c t i v e member o f the s e r i e s . E l e c t r o n donating groups f o r R , e.g., NH (Compound 38) and OMe (Compound 39), produced a c o n s i d e r a b l e decrease i n a c t i v i t y . I t i s remarkable that when the other hydrogen atom (R_) was s u b s t i tuted by CI, as i n compounds 40 and 41, a c t i v i t y was t o t a l l y l o s t . Some v a r i a n t s o f R induce the pyridone r i n g I to assume i t s tautomeric form I I (Figure 4 ) . I f R i s an oxygen atom, the pyridone c o n f i g u r a t i o n I dominated almost completely, as confirmed by IR data. T h i s was a l s o the case with compound 43 and probably with compound 42, but i f R =0Me (Compound 44) or CI (Compound 46), only tautomer I I i s p o s s i b l e . Since among the compounds which e x i s t i n one or the other tautomeric form, both a c t i v e as w e l l as i n a c t i v e compounds are found (Table I V ) , the a c t u a l r i n g s t r u c t u r e would appear not to be r e l e v a n t . 1 0

1Q

1Q

2

q

g

Q

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

144

Table IV. E f f e c t o f v a r i a t i o n o f ife

i

AGENTS

n

COOEt EtO-CHg no.

Re

1

OH

ED o

3-8 toi?

SH NH kk

no.

20

2

OMe

to

13 30

ED200 (mg/kg)

Re NHPr

h6

CI

Γ J

Ο Γ

· · inactive

The SAR may be summarized by the statement that the t r i c y c l i c compound must be l i n e a r and unsubstituted a t p o s i t i o n s 2, 5, 6 and 7. S u b s t i t u t i o n at p o s i t i o n 3 with a hydroxymethyl or alkoxymethyl group i s necessary, while a t p o s i t i o n 8, a s u b s t i t u e n t such as a carboxyl, alkoxycarbonyl, hydroxyalkyl, a c e t y l , or a l k y l group must be present. The oxygen atom normally present a t p o s i t i o n 9 may be replaced by c e r t a i n groups, and s u b s t i t u t i o n a t p o s i t i o n 10 by l i p o p h i l i c e l e c t r o n e g a t i v e groups increases the a c t i v i t y . A QSAR f o r s u b s t i t u e n t s at p o s i t i o n 10 confirmed these statements. I n the c a l c u l a t i o n s concerning RQ, three d e v i a t i o n s from the r e g r e s s i o n were found, i . e . , with compounds 28, 29 and 31. PHYSICOCHEMICAL PROPERTIES The compounds o f t h i s s e r i e s possess an extremely low s o l u ­ b i l i t y i n water (Table V). The p a r t i t i o n c o e f f i c i e n t s i n d i c a t e d a r a t h e r l i p o p h i l i c character, but the s o l u b i l i t y i n l i p o p h i l i c solvents was g e n e r a l l y very low, being f a r l e s s than 0.1$ i n most s o l v e n t s . The e s t e r , quincarbate (Compound 33), e x h i b i t s good s o l u b i l i t y (>10$) only i n s t r o n g l y protonating s o l v e n t s , while the a c i d s (Compounds 47 and 51) form s o l u b l e s a l t s i n aqueous a l k a l i .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9.

BOSCHMAN ET AL.

Table V.

Quincarbate

145

Comparison of s o l u b i l i t y and p a r t i t i o n c o e f f i c i e n t with diuretic a c t i v i t y i n rats Solubility

Partition

Diuretic

i n water

coeffi-

activity

cient

ED oo

at 20°C

octanol/

(mg/kg)

(mg/L)

water

at pH COOR

6

at pH No.

Re

Rio

R Et

30 1

1

EtOCHg

H

33 47

EtOCHg

Cl

Et

EtOCH

2

Cl

H

48

EtOCH

2

Cl

CI^-CHgOH

49 50 51 52

EtOCHs

Cl

CH -CH0H-CH 0H:

EtOCH

Cl

CHg-0-COMe

Cl

H

Cl

Et

H0CH

2

HOCHg

2

2

2

7-5

3-8

> 50 > 50

0.08

10 2

20

4.5

0.5 1.0

1.6

1.5 30

1.7 5.7

~1 /o0.1

Since the extremely low s o l u b i l i t y (<30 mg/L) of the compounds might i n f l u e n c e the b i o a v a i l a b i l i t y , some compounds were synthes i z e d with more h y d r o p h i l i c s u b s t i t u e n t s (Table V, Compounds 48, 49, 51 and 52). However, no improvement i n a c t i v i t y was accomplished. I t may be concluded that, i n s p i t e of the r o l e played by the l i p o p h i l i c c h a r a c t e r of c e r t a i n groups (e.g., CI at R ) , the l i p o p h i l i c i t y o f the whole molecule ( p a r t i t i o n c o e f f i c i e n t ) does not seem to be an important f a c t o r i n the l e v e l of a c t i v i t y t h a t i s achieved (compare compounds 33 and 47 with 52, and compound 1 with 3 3 ) · A l s o , an extremely low s o l u b i l i t y does not seem to have a negative e f f e c t on the a c t i v i t y i n the r a t . 1 Q

PHARMACOLOGICAL PROFILE Quincarbate (Compound 33, F i g u r e 5) was s e l e c t e d f o r development as a p o s s i b l e drug f o r human therapy. The v a r i o u s e x i s t i n g d i u r e t i c s can be d i v i d e d i n t o two c l a s s e s : "high c e i l i n g " d i u r e t i c s , of which furosemide i s a well-known r e p r e s e n t a t i v e , and "low c e i l i n g " d i u r e t i c s , a c l a s s to which most d i u r e t i c s of the t h i a z i d e type belong. The d i f f e r e n c e can be demonstrated most c l e a r l y i n s a l i n e - l o a d e d r a t s using the L i p s c h i t z method (2). Using t h i s procedure, quincarbate was shown to be a "high c e i l i n g " d i u r e t i c with an e f f i c a c y comparable to that of furosemide

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

146

volume

AGENTS

(pooled over 5 h r s )

ml/kg

^

\

%

Λ

/

60

9

40

quincarbate

Δ

furosemide

•

chlorothiazide control

20

level

^ •-

dose 0.01 Figure 6.

0.1

1.0

10.0

(oral)

100 mg/kg

Dose-response rehtionships in male albino rats (strain Wistar-TNO, 160 ± 20 g), six animah per dose group

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9.

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Quincarbate

147

(Figure 6 ) . S i g n i f i c a n t d i u r e t i c e f f e c t s of quincarbate i n r a t s are e l i c i t e d at doses which are at l e a s t 200 times lower than the minimum dose r e q u i r e d f o r furosemide and c h l o r o t h i a z i d e . However, s i n c e the dose/response curves do not run p a r a l l e l , a s t r i c t comparison o f the potencies i s not p o s s i b l e . The e l e c t r o l y t e e x c r e t i o n caused by quincarbate, furosemide and c h l o r o t h i a z i d e , r e s p e c t i v e l y , p a r a l l e l s t h e i r d i u r e t i c r e sponse (Figure 7 ) . Over the complete dose range, quincarbate gives a more f a v o r a b l e r a t i o of the Na /K e x c r e t i o n when compared to furosemide, and i t i s even b e t t e r when compared to c h l o r o t h i a z i d e (Figure 8 ) . The dehydration of the animals caused by e f f e c t i v e d i u r e t i c s masks t h e i r e f f e c t i v e n e s s over longer time i n t e r v a l s . In order to prevent such an i n t e r f e r e n c e i n the study of the d u r a t i o n of a c t i o n , the r a t s were i n t e r m i t t e n t l loaded with s a l i n i volume equal to the volum p e r i o d . From F i g u r e 9 onset of a c t i o n , as does furosemide. However, the a c t i o n cont i n u e s over a p e r i o d of seven hours, a f t e r which i t g r a d u a l l y subsides, whereas furosemide has a much s h o r t e r d u r a t i o n of action. When t e s t e d i n r a t s not loaded with s a l i n e at a d a i l y dose of 0.1 mg/kg f o r 14 days, the d i u r e t i c a c t i v i t y of quincarbate remained undiminished. No rebound e f f e c t s were observed a f t e r d i s c o n t i n u i n g the a d m i n i s t r a t i o n of the drug. A l s o , i n waterdeprived r a t s , a d i u r e t i c e f f e c t was c l e a r l y seen. Quincarbate shows a remarkable v a r i a t i o n i n i t s d i u r e t i c e f f e c t i v e n e s s i n v a r i o u s s p e c i e s . In dogs, an ED^QQ value of 5 mg/kg o r a l l y was obtained. I t i s noteworthy t h a t i n t h i s s p e c i e s the n a t r i u r e t i c response was more pronounced than the d i u r e t i c response ( F i g u r e 10). S u r p r i s i n g l y , i n mice an a n t i d i u r e t i c e f f e c t was noted at low doses (1 mg/kg), whereas, at higher doses (46 and 100 mg/kg), some d i u r e t i c e f f e c t was apparent. In hams t e r s , quincarbate e x h i b i t e d only marginal a c t i v i t y . From both i n v i t r o and i n v i v o s t u d i e s , i t can be concluded that quincarbate i s not a carbonic anhydrase i n h i b i t o r . It exerted i t s d i u r e t i c e f f e c t even i n drug-induced a c i d o t i c r a t s . The potency was u n a f f e c t e d i n adrenalectomized r a t s ; furthermore, no d i r e c t aldosterone antagonism was observed. In r a t s with a r t i f i c i a l l y induced edema ( p a r t i a l hepatectomy), quincarbate proved to be h i g h l y e f f e c t i v e . Quincarbate was t e s t e d f o r a n t i h y p e r t e n s i v e a c t i v i t y i n spontaneously hypertensive (SH) r a t s (3). The drug was admini s t e r e d o r a l l y , f o l l o w e d 20 hours l a t e r by a second dose; then 4 hours l a t e r , the blood pressure was measured by a cannula i n the caudal a r t e r y . A s l i g h t a c t i v i t y was observed at 1 mg/kg, and a maximum e f f e c t was seen at 5 mg/kg. A prolonged a c t i o n was i n d i c a t e d by n o t i n g t h a t the o r i g i n a l blood pressure was somewhat decreased even 20 hours a f t e r the f i r s t dose. With a dose of 5 mg/kg, the a c t u a l lowering ( i n mm Hg) was from 169.0 +9.6 to +

+

American Chemlcaf

Society Library 1155 16th St. N. W. In Diuretic Agents; Cragoe, E.; Washington, D. C. Society: 20031Washington, DC, 1978. ACS Symposium Series; American Chemical

DIURETIC

148

AGENTS

Excretion meq/kg quincarbate ο"

3

ira 0.01

[1 0*033

0.10

Q=Na

1

0.33

1.0

I-

dose(oral)

3-3

12.5

mg/kg

i

furosemid

10

33

100

330

chlorothiazide

0.33

1.0

3.3

10

100

Figure 7. Dose-response of electrolyte excretion in saline-loaded rats. Six animals per dose group were used. Determinations of electrolytes were performed in pooled urine samples excreted in the five-hour period.

Figure 8.

Ratios of Na*:K* excretion in saline-loaded rats. (Further details in legend of Figure 7.)

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9. BOSCHMAN ET AL.

Quincarbate

149

138.3 + 7.3 a t the f o u r t h hour on the second day (3). I n r e n a l hypertensive r a t s , no c o n c l u s i v e e f f e c t s were found with e i t h e r quincarbate or furosemide. Following o r a l a d m i n i s t r a t i o n o f quincarbate to r a t s a t doses o f up to 100 mg/kg, no t o x i c o r neurotoxic phenomena were observed. A l s o , i n c h r o n i c t o x i c i t y s t u d i e s with r a t s and dogs, no s e r i o u s s i d e e f f e c t s were observed at doses o f 50 mg/kg. In r a b b i t s and r a t s , quincarbate d i d not adversely a f f e c t pregnancy, nor d i d i t show t e r a t o g e n i c p o t e n t i a l . METABOLISM The metabolism o f quincarbate was s t u d i e d i n r a t s , dogs, monkeys and humans by means o f C - l a b e l l e d m a t e r i a l . From a number o f experiments i n normal r a t s as w e l l as i n r a t s with cannulated or l i g a t e d b i l duct it b deduced that th r a t i of u r i n a r y to b i l i a r y e x c r e t i o 2:1. The m a t e r i a l that enterohepatic c i r c u l a t i o n but i s excreted i n the f e c e s . Using the above-mentioned r a t i o , we c a l c u l a t e d the amount o f absorption on the b a s i s o f the u r i n a r y e x c r e t i o n o f r a d i o a c t i v e m a t e r i a l . I n a l l s p e c i e s , the degree o f a b s o r p t i o n was s t r o n g l y dose dependent. In r a t s , the degree o f a b s o r p t i o n decreased from 45Î a t a dose o f 1 mg/kg to 15% a t the dose o f 16 mg/kg. I n dogs, the absorption percentage d e c l i n e d from 40? a t the dose o f 0.1 mg/kg to 2.25? a t 50 mg/kg. I n monkeys, a t a dose o f 1 mg/kg, the absorption was 10?. In humans, the degree o f absorption d e c l i n e d from 60? a t 0.015 mg/kg t o 55? a t 0.075 mg/kg and f i n a l l y t o 44$ at 0.3 mg/kg. Hence, w i t h i n the t h e r a p e u t i c range, about 50? o f the dose w i l l be absorbed. U r i n a r y e x c r e t i o n i n a l l species ended w i t h i n 24 hours. Plasma l e v e l s o f r a d i o a c t i v e m a t e r i a l were extremely low a t a l l times i n r a t s , dogs and humans. Maximum l e v e l s tended to be reached w i t h i n one to three hours. N e i t h e r unchanged quincarbate nor the corresponding a c i d , nor even conjugates, were found i n the e x c r e t i o n products. In the urine o f the r a t and the monkey, seven metabolites could be i d e n t i f i e d . In dogs and man, only three o f these were found. Of these three, the one which occurred i n the major p r o p o r t i o n by f a r proved to be compound 51 (Figure 11). Thus, both ether and e s t e r h y d r o l y s i s must have occurred. Since both the hydroxymethyl d e r i v a t i v e of the a c i d , compound 51, as w e l l as o f the corresponding e s t e r , compound 52, show high a c t i v i t y (Table V ) , these products may a l s o play an important r o l e i n the observed d i u r e t i c e f f e c t . Since one o f the c h a r a c t e r i s t i c s o f quincarbate i s i t s species dependent d i u r e t i c a c t i v i t y , a study was conducted to r e v e a l whether o r not t h i s was a matter o f metabolism. The d i u r e t i c a c t i v i t y o f compound 52, the e s t e r o f the main metabolite, was studied i n various s p e c i e s . I n dogs, compound 52 showed an a c t i v i t y comparable to that o f quincarbate, and, i n mice, i t was completely i n a c t i v e a t doses up t o 10 mg/kg. Therefore,

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

150 Excretion

AGENTS

(urine)

ml/kg

20

10

•

quincarbate 0. 2 mg/kg

S

furosemide 25 mg/kg

Π control

10 h r s Figure 9. Duration tration. Six animals per group were used. Rats were intermittently loaded with saline. Excretion o f mean c o n t r o l

400

300

200 ο volume χ Na

100

1

dose

7-5

2.5

22.5

mg/kg

Figure 10. Urine and Na excretion in beagle dogs after oral administration of quincarbate. Seven dogs per dose group were used. Excreted urine was pooled after seven hours. +

COOR Figure 11. Main excretion product of quin­ carbate and its ester. 51: R = H. 52: R = HO-CH, Et.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9.

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151

Quincarbate

i t i s u n l i k e l y that the species dependence i s due to d i f f e r e n t degrees o f formation o f the a c t i v e m e t a b o l i t e s . I t i s possible that the degree o f absorption o f quincarbate i n the v a r i o u s s p e c i e s determines, to a c e r t a i n extent, the v a r i a t i o n i n d i u r e t i c potency. HUMAN PHARMACOLOGY The e f f e c t o f quincarbate on the e x c r e t i o n o f u r i n e and o f u r i n a r y e l e c t r o l y t e s has been i n v e s t i g a t e d i n s i x small s t u d i e s (4-8), i n c l u d i n g 34 healthy v o l u n t e e r s . In a l l these s t u d i e s , quincarbate was given as a s i n g l e o r a l dose, but s e v e r a l s u b j e c t s i n g e s t e d the drug i n d i f f e r e n t dosages on subsequent t e s t days. T h i s r e s u l t e d i n a t o t a l o f 84 s i n g l e - d o s e a d m i n i s t r a t i o n s a t d i f f e r e n t dose l e v e l s . Th f o l l o w i n p r e l i m i n a r conclusion be drawn: Potency. I n the range o f 5 t o 40 mg, the dose/response curve o f quincarbate i s very f l a t . T h i s might be p a r t i a l l y explained by incomplete a b s o r p t i o n a t the higher doses i n man. Quincarbate proved t o be q u i t e potent; thus, 10 t o 20 mg given o r a l l y showed n a t r i u r e t i c e f f e c t s comparable with those o f 100 mg hydrochloro­ t h i a z i d e (4) (Table V I ) . Table V I .

D i u r e t i c response i n humans t o quincarbate and h y d r o c h l o r o t h i a z i d e (4·) ( d o u b l e - b l i n d study i n v o l v i n g s i x h e a l t h y male v o l u n t e e r s between the ages o f 22 and 32 y e a r s ) . Mean Cumulative Response Diuresis (ml) after 12 h r s

Placebo

+

Natriuresis (meq)

after hrs

after 12 h r s

695

1218

6l

126

2.0

2.7

978 1162 1082 1315

1598 1903 1998 2217

145

239 266 309

3Λ

k.o

152 170 158

3-7

2jk

2.9 3Λ 2.5

3.2

1223

2110

I83

310

4-.2

4.9

24-

after

Νβ /Κ* ratio

after hrs

24· h r s 12

after hrs

24·

quincarbate

5 10 20

mg mg mg

kO mg

k.i

hydrochloro­ thiazide

100

mg

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

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AGENTS

The o r a l a c t i v i t y of quincarbate was compared i n four s t u d i e s (4-6, 8) with that of furosemide. While 5 (or 20) mg of q u i n carbate proved to be more n a t r i u r e t i c than 20-25 mg of furosemide, 40 mg of furosemide sometimes showed a l e s s e r and sometimes a g r e a t e r e f f e c t than 20 mg of quincarbate. With 80 mg of f u r o semide, greater n a t r i u r e s i s and d i u r e s i s could be achieved than with quincarbate. Table VII gives the r e s u l t s of one of these studies. 1

Pattern of A c t i v i t y . Quincarbate s d i u r e t i c a c t i v i t y s t a r t s w i t h i n one hour a f t e r i t s o r a l i n g e s t i o n and l a s t s f o r at l e a s t 8-9 hours, but (as can be i n f e r r e d from Table VI) d i u r e s i s and n a t r i u r e s i s continue even during the 12-24 hour i n t e r v a l . Table V I I .

D i u r e t i c response i n humans t o quincarbate and furosemide cross-ove female healthy v o l u n t e e r s between the ages o f 22 and 6 l y e a r s ) . Mean cumulative response a f t e r 8 hrs

Placebo

NaVs* ratio

Natriuresis (meq)

Diuresis (ml)

608

40.8

2.0

1232 1262 1204

137.3 143.9 142.4

4.6 3.5 3.9

1214 1772

117.2 180.8

3-3 4.2

quincarbate

5 10 20

mg mg mg

furosemide

20 40

mg mg

+

While there i s an i n c r e a s e i n u r i n a r y K e x c r e t i o n , i t i s l e s s than the corresponding i n c r e a s e i n u r i n a r y Na e x c r e t i o n . T h i s r e s u l t s i n a f a v o r a b l e Na /K r a t i o , comparable to that f o l l o w i n g i n g e s t i o n of furosemide. Both the d i u r e t i c and n a t r i u r e t i c response are a l s o w e l l maintained i n dehydrated s u b j e c t s . The main anion excreted c o n c u r r e n t l y with the i n c r e a s e i n Na and K e x c r e t i o n i s the c h l o r i d e i o n . There i s no i n d i c a t i o n o f any i n h i b i t i o n of tubular bicarbonate r e a b s o r p t i o n . N e i t h e r the glomerular f i l t r a t i o n r a t e (as measured by the r a t e o f i n u l i n clearance) nor the r e n a l plasma flow (as measured by the paminohippuric a c i d clearance) are a f f e c t e d by quincarbate. +

+

+

+

Side E f f e c t s and Tolerance.

Up to the present time, quincarbate

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

9.

BOSCHMAN ET AL.

153

Quincarbate

has been t o l e r a t e d extremely w e l l ; no c l i n i c a l s i d e e f f e c t s of any importance have been observed. O c c a s i o n a l l y , a s l i g h t decrease i n serum N a and/or K was noted, as might be expected with any potent d i u r e t i c . +

+

CINICAL STUDIES 1

Our c l i n i c a l s t u d i e s to assess q u i n c a r b a t e s e f f i c a c y and s i d e e f f e c t s are i n t h e i r e a r l y stages. Only one (j>) s m a l l doubleb l i n d cross-over study i n h o s p i t a l i z e d p a t i e n t s with p e r i p h e r a l c a r d i a c edema has been completed. In t h i s study, c o n s i d e r a b l e improvement of c l i n i c a l edema was apparent i n a l l p a t i e n t s . In the cross-over study, an o r a l dose of 10 mg quincarbate was compared with 50 mg h y d r o c h l o r o t h i a z i d e ; the o v e r a l l response to both drugs (improvement u r i n e and Na , f a v o r a b l l y t e s ) was q u i t e s i m i l a r . +

CONCLUSIONS (1) Some d i o x i n o ^ , 3 r g 7 q u i n o l i n e d e r i v a t i v e s e x h i b i t extremely potent d i u r e t i c a c t i v i t y with a high c e i l i n g c h a r a c t e r i n the r a t . (2) The s t r u c t u r e s of t h i s group d i f f e r markedly from those of e s t a b l i s h e d d i u r e t i c s . (3) The s t r u c t u r a l v a r i a t i o n s p e r m i s s i b l e f o r d i u r e t i c a c t i v i t y are very l i m i t e d . (4) The most a c t i v e compound, quincarbate ( F i g u r e 5), which at an o r a l dose of 0.08 mg/kg doubled the volume of the u r i n e excreted by the r a t , was s t u d i e d e x t e n s i v e l y . (5) Upon repeated a d m i n i s t r a t i o n of quincarbate to r a t s , no t o l e r a n c e or rebound e f f e c t s were noted. (6) The d i u r e t i c a c t i v i t y of quincarbate was s p e c i e s dependent; i t e x h i b i t e d potent a c t i v i t y i n r a t s and humans, moderate a c t i v i t y i n dogs and monkeys and only marginal a c t i v i t y i n mice and hamsters. (7) The main metabolite of quincarbate (compound 51) a l s o possessed d i u r e t i c a c t i v i t y . (8) In c h r o n i c t o x i c i t y s t u d i e s i n r a t s and dogs, no notable t o x i c e f f e c t s were observed at o r a l doses up to 50 mg/kg. (9) In healthy humans, an o r a l dose of 5 mg of quincarbate showed a marked d i u r e t i c and n a t r i u r e t i c e f f e c t ; and yet, even a t the h i g h e s t dose used (20 mg), no s i d e e f f e c t s of any importance were noted. (10) In p a t i e n t s with p e r i p h e r a l c a r d i a c edema, a cons i d e r a b l e improvement of the edema was apparent with low doses (10 mg o r a l l y ) . However, f u r t h e r s t u d i e s w i l l be r e q u i r e d before quincarbate can be assigned i t s place among f u t u r e t h e r a p e u t i c s . Acknowledgments - We acknowledge the e f f o r t s of a l l our c o l l e a g u e s i n the r e s e a r c h l a b o r a t o r i e s of Philips-Duphar who have

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

154

i n v o l v e d i n the s t u d i e s which we have d e s c r i b e d . We mention e s p e c i a l l y K. S. Liem, M.D., who coordinated the s t u d i e s i n man, Dr. J. B. v.d. Schoot, who c a r r i e d out the metabolic s t u d i e s , and Mr. J. T i p k e r , who provided the v a l u a b l e QSAR c a l c u l a t i o n s .

Literature Cited 1. Dijk, J. van, Hartog, J. and Boschman, T h . A . C . , J. Med. Chem. (1976), 19, 982. 2. Lipschitz, W. L., Hadidian, Z. and Kerpscar, Α . , J. Pharmacol. Exp. Ther. (1943), 92, 97. 3. Denton, J. J., Lederle Labs., U.S.A., Private Communication. 4. Uhlich, E., Univ. Munich, Germany, Private Communication. 5. Mitchell, G. Μ., Cardiff, U . K . , Private Communication. 6. Hitzenberger, G . , Univ. Vienna, Austria, (2 studies), Private Communications. 7. Vrhovac, B . , Univ. Zagreb Yugoslavia Private Communication 8. Wayjen, R. G. A. va Netherlands, Private Communication RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

10 Etozolin: A Novel Diuretic G. SATZINGER, M . HERRMANN, K.-O. VOLLMER, A. MERZWEILER, and H . GOMAHR Goedecke Research Institute, Goedecke AG, D-78 Freiburg (in Breisgau), West Germany O. HEIDENREICH and J. GREVEN Pharmacological Department, Rhein.-Westf. Technical University of Aachen, West Germany

Introduction - Investigations to date seem to indicate that choleresis i s , to some extent, part of the extrarenal effect of diuretic substances. However, more detailed studies, p a r t i c ularly with furosemide and ethacrynic acid, have proved that b i l i a r y osmolality i s not affected hereby (1-4). The question as to whether choleretics, which do not influence the b i l i a r y electrolyte concentration, would make suitable models in the search for novel diuretic substances has never been investigated. We have recently described (5) some highly potent diuretic and well-tolerated compounds which were discovered in the course of investigating a new class of heterocycles (Structure 1), many of which possess choleretic properties.

One of these compounds, ethyl Z-[3-methyl-4-oxo-5-(1-piperidinyl)-2-thiazolidinylidene]acetate (Compound 3, etozolin), has been introduced as a therapeutic agent on the German market under the trademark Elkapin . Table I summarizes the current synthetic work i n the f i e l d of 2-acylmethylene-4-thiazolidinones. Table II shows the substitution pattern of the compounds of type 1 which are active diuretics. As a consequence of these findings, six other series of heterocycles were synthesized and tested (Table III). None of the representatives of these heterocycles exhibited diuretic a c t i v i t y , even when the successful substitution pattern shown in Table II was employed (as far as this was feasible and led to stable compounds). R

0-8412-0464-0/78/47-083-155$09.00/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

156

AGENTS

TABLE I Synthesized and Tested D e r i v a t i v e s of 1

Substituent

Notes

Groups H, a l k y l , a r a l k y l , (CH ) X 2

CH X,

X = f u n c t i o n a l groups such as -OR, -NR 2>

2

H, a l k y l , a r y l , a r a l k y l , CH X, ( C H ) X ; NHCOCH ; 2

2

2

3

C0 R

only f o r R

= CH„

CH

only f o r R

= C0 R

2

3

2

-C0 H, -C0 R, R-CO-, 2

2

ArCO-, R-S0 ~, A r S 0 ~ , 2

2

>NCO-, -HNCO-, R NC0-,

AR = a r y l , h e t e r o aryl

2

-CN; p y r i d y l , benzimidazolyl, indolyl H, a l k y l ,

RV

aryl 2

>CR

-N<, -N-N<

a l i p h . , arom., aliph.-arom., heteroa l i p h . and arom. amines and hydrazines

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

10.

Etozolin: A Novel Diuretic

SATZINGER ET AL.

157

TABLE I I D e r i v a t i v e s of 1 and T h e i r D i u r e t i c R e l a t i v e to E t o z o l i n (3)

Compound No.

1

2

1

R

R

H

H

CH

3

H

CH

3

H

CH

3

CH

3

R

C 0

C

U

J

C H

R

-0 0 I-

I

l.

Λ

H

2 2 5

2

S

R

C0

C 0

Activity

3

- o

H

C0 H

- O

Η

C0 H

2

2

Diuretic Activity ( R e l a t i v e to 3)

°-

H

H

3

1-1

J , I

Η

1.3

L

Η

0.6

| CE,

C H

3

8

CH

9

CH

3

H

C 0

CH

C

C 0 3

u u

2 C H 2

10

CH

3

Η

11

CH

3

H

12

CH

3

H

H

2 2 5

C

H

2 2 5

C

0SO

C H

H

2 2 5

3

2

S 0

2

J

'

I

CH 5

C 0

-Ο -Ο -Ο -Ο -Ο -Ο

-k

Ji

C H

3

H

J,

I

Γ

I

1

°-

I

\

°-

Η

0.6

°'

H

Η

Η

7

6

0.5

0.3

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

158

TABLE I I I Schematic Representation of Heterocycles S t r u c t u r a l l y Related to 1

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

10.

SATZINGER ET

AL.

Etozolin: A Novel Diuretic

159

SAR o f 2-Acylmethylene-4-Thiazolidinones - Changes i n the s u b s t i t u e n t on the r i n g n i t r o g e n (R ) o f compound 1 markedly a f f e c t d i u r e t i c a c t i v i t y . Even the change o f R = CH^ to ^Hpleads to |n abrupt l o s s of a c t i v i t y . Almost the sam^can be s a i d of R because a c t i v i t y i s maintained only when R = H or CH~. Stereochemistry does not play an important r o l e here s i n c e both the Z- and the Ε-isomer are a c t i v e . Maximal a c t i v i t y i s seen when R"* i s C00H or COOEt; the only other s u b s t i t u e n t s allowed are methyl and p h e n y l s u l f o n y l , and they produce l e s s ^ a c t i v e compounds. Optimal a c t i v i t y i s c l e a r l y achieved when R i s 1 - p i p e r i d y l . Any enlargement or r e d u c t i o n of the r i n g s i z e or any b r i d g i n g or s u b s t i t u t i o n reduces the a c t i v i t y and 2,6d i s u b s t i t u t i o n e l i m i n a t e s the a c t i v i t y . I n t r o d u c t i o n o f a second s u b s t i t u e n t a t C-5 ( i . e . , where R i s not H) does not lead to a c t i v e compounds A particularly fascinatin most potent d i u r e t i c an ς

However, as i s i l l u s t r a t e d below, compound 3 possesses only a very weak c h o l e r e t i c ( s i d e ) e f f e c t and compound 13 has no d i u r e t i c e f f e c t . We i n v e s t i g a t e d t h i s phenomenon u s i n g spec­ t r o s c o p i c , p h y s i c a l chemical and X-ray measurements. The s p e c t r o s c o p i c and thermodynamic f i n d i n g s have been published (5), and some of the r e s u l t s o f the X-ray s t u d i e s w i l l be discussed subsequently. Comparison of 2-Acylmethylene-4-Thiazolidinones with Other C l a s s e s o f D i u r e t i c s - As i t w i l l be discussed i n d e t a i l below, the 2-acylmethylene-4-thiazolidinones represent a new c l a s s o f h i g h - c e i l i n g d i u r e t i c s to be added to the w e l l known sulfamyl benzoic a c i d s and acylphenoxyacetic a c i d s . They a l l seem to have a s i m i l a r e f f e c t on r e n a l e l e c t r o l y t e t r a n s p o r t . An examination o f e t o z o l i n , furosemide and e t h a c r y n i c a c i d , each a r e p r e s e n t a t i v e o f one o f the three d i u r e t i c c l a s s e s , r e v e a l s that they a l l have (a) the same main s i t e o f a c t i o n ( e a r l y d i s t a l t u b u l a r s i t e ) and (b) presumably, the same mechanism o f a c t i o n ( i n h i b i t i o n o f a c t i v e c h l o r i d e t r a n s p o r t ) (.6-9.). At the present time, l i t t l e i s known about the mechanism o f a c t i o n of these d i u r e t i c s ; there are some i n d i c a t i o n s that i t i n v o l v e s i n h i b i t i o n of a r e n a l adenyl c y c l a s e (10). In order to develop working hypotheses on which to base an economical search

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

160

AGENTS

f o r new d i u r e t i c s , i t was o f i n t e r e s t to i n v e s t i g a t e whether these c h e m i c a l l y d i f f e r e n t substances had some common s t r u c t u r a l and/or e l e c t r o n i c f e a t u r e s . Such an approach seemed reasonable c o n s i d e r i n g the molecular r i g i d i t y o f these c l a s s e s o f compounds. Figure 1 shows on the l e f t the three c l a s s e s o f h i g h - c e i l i n g d i u r e t i c s : acylmethylene-4-thiazolidinones (14), sulfamoylbenzoic a c i d s (15) and acylphenoxyacetic a c i d s (16). The s t r u c t u r a l f e a t u r e s thought to be important f o r a c t i v i t y are d e l i n e a t e d i n the f i g u r e (11). On the r i g h t , the e l e c t r o n i c e f f e c t s o f these f u n c t i o n a l groups are i n d i c a t e d by the arrows; the length and d i r e c t i o n o f the arrows r e f l e c t t h e i r r e l a t i v e e l e c t r o n donor and acceptor p r o p e r t i e s . A s t r i k i n g f e a t u r e i s the general p a t t e r n that appears whose dominant c h a r a c t e r i s t i c s are two groups with opposing e l e c t r o n i c e f f e c t s attached raetaor para- to each other and l i n k e d by a r i n g system s u i t a b l e f o r the t r a n s m i s s i o n o f t h e i between comparable charg furosemide, N /S; e t h a c r y n i c a c i d , l-0/=C-C=0) are i n the range of 5.5-6.0 A. These d i s t a n c e s were determined f o r compound 3, from X-ray data, see below; furosemide, c a l c u l a t e d from the data of P r i c e (12) and Dupont (13), e t h a c r y n i c a c i d data from D r e i d i n g models. X-ray a n a l y s i s o f compound 3 ( d e t a i l e d data on comp a r a t i v e X-ray a n a l y s i s o f the acylmethylene-4-thiazolidinones w i l l be published i n a subsequent paper) f u r n i s h e s i n t e r e s t i n g information ( F i g u r e 2 ) . The h e t e r o c y c l e , i n c l u d i n g the s t r u c t u r a l fragment shown below, i s v i r t u a l l y p l a n a r . The symmetry a x i s N / C - l l o f the p i p e r i d i n e r i n g i s approximately e q u a t o r i a l ar

to t h i s plane, and the averaged plane of the p i p e r i d i n e c h a i r i s roughly v e r t i c a l to i t . The i n t e r a t o m i c d i s t a n c e s (Table IV) subs t a n t i a t e the i n c l u s i o n o f the r i n g s u l f u r S i n the e l e c t r o n d i s t r i b u t i o n o f the whole p l a n a r system. In accordance with i t s e l e c t r o n i c s i t u a t i o n and i n c o n t r a s t to the enone double bond o f the e t h a c r y n i c a c i d (19), the e x o c y c l i c double bond i s not n u c l e o p h i l i c ; compound 3 does not r e a c t with s u l f h y d r y l groups. T h i s f a c t does not c o n t r a d i c t our argumentation because the d i u r e t i c potency o f the acylphenoxyacetic a c i d s do not run p a r a l l e l to the n u c l e o p h i l i c c h a r a c t e r of t h i s bond (20). A n a l y s i s o f the X-ray data f u r n i s h i n g some explanations f o r the unusual SARs of the acylmethylene-4-thiazolidinones i s as

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

SATZINGER E T

AL.

Etozolin: A Novel Diuretic

Figure 1. The three classes of high-ceiling diuretics with the moieties responsible for biological activity and their effects on electron distribution

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

162

Numbering

JO

3

o 8 7 2 || CH,-CH -0-C 3 2 \ ή

o

( - £ i ^ - o N' 5

/

1

U

Figure 2. Dreiding Model of Compound 3 constructed on the basis of x-ray data. Viewed along the Z-axis (N : (0,0,0)). ( The Dreiding model simulâtes plananty for the entire hetero ring not considering the deviation of S' by about 5°; thus, erroneously, 3-H is nearly eclipsed by C3-N2.) t

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

10.

SATZINGER E T A L .

Etozolin: A Novel Diuretic

163

TABLE IV C h a r a c t e r i s t i c Interatomic Distances and Bond Angles of Compound 3 and t h e i r A n t i c i p a t e d Values

E t o z o l i n (3) $)

Comparable Standard Values

(£)

Ref.

N2-C3

1.43

C3-S1

1.89

1.82 ( a l i p h a t i c )

14

Sl-Cl

1.74

1.82 ( a l i p h a t i c ) 1.72 (thiophene)

14 15

C1-C5

1.36

1.30 ( a c r y l i c a c i d )

16

C5-C6

1.41

1.47 ( a c r y l i c a c i d )

16

C6-03

1.24

1.26 ( a c r y l i c a c i d )

16

C6-02

1.34

1.28 ( a c r y l i c a c i d )

16

92°

91.9° Cthiophene 100-105° ( a l i p h a t i c ) 105.5° ( t h i a z o l i d i n e )

15 17 18


In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

164

AGENTS

1

f o l l o w s : 1) the exchange o f the r i n g s u l f u r S"" f o r other heteroatoms o r a change i n i t s o x i d a t i o n s t a t e should a f f e c t or reduce i t s pseudo-aromatic s t a t e , 2) any i n c r e a s e i n the s i z e of the s u b s t i t u e n t on the r i n g n i t r o g e n beyond CH^ should a l t e r the e l e c t r o n d i s t r i b u t i o n i n the molecule due t o d i s t u r b a n c e o f p l a n a r i t y o r o f hyperconjugation; the same a p p l i e s t o the s t e r i c bulk and the nature o f the acylmethylene^group and 3) i n t e r ­ a c t i o n between the p i p e r i d i n o n i t r o g e n Ν and the h e t e r o c y c l i c r i n g i s dependent on the former's unhindered s p a t i a l f l e x i ­ bility. Other i n f l u e n c e s a r e probably a t t r i b u t a b l e t o the e f f e c t s o f the 5-amino group on t r a n s p o r t ( 5 ) . PHARMACOLOGY Acute T o x i c i t y - Th i n male mice (NMRI) an LD values was based on the p r o b i t a n a l y s i s a c c o r d i n g t o Weber (21;. The r e s u l t s a r e shown i n Table V. Q

TABLE V Acute T o x i c i t y o f E t o z o l i n i n Mice and Rats Following i . g . and i . p . A d m i n i s t r a t i o n

Species

Sex

Route o f Adminis­ tration

L D

50 (mg/kg)

Confidence L i m i t s ρ = 0.05 (mg/kg) lower upper

Mouse

male

i.g.

8,670

7,310

10,270

Mouse

female

i.g.

9,360

7,290

12,030

Mouse

male

i.p.

1,210

1,093

1,340

Rat

male

i.g.

11,040

9,380

13,000

Rat

female

i.g.

10,250

9,260

11,350

Rat

male

i.p.

1,575

1,355

1,830

As the values shown i n Table V i n d i c a t e , the acute i . g . and i . p . t o x i c i t y o f e t o z o l i n i s extremely low. The LD_ values d i d not show any a p p r e c i a b l e d i f f e r e n c e i n male or female animals. The t o x i c i t y o f the compound i s s l i g h t l y l e s s i n r a t s than i n mice. The low p.o. t o x i c i t y o f the drug was a l s o confirmed i n a p i l o t dose r a n g e - f i n d i n g study i n dogs. In t h i s s p e c i e s , the f i r s t evidence o f t o x i c i t y occurred a t 1,600 mg/kg; a t 3,200 mg/kg, the lower l e t h a l dose range was reached. D i u r e t i c A c t i v i t y - Clearance T e s t s i n Dogs: These s t u d i e s served t o r e v e a l the d i u r e t i c e f f e c t o f e t o z o l i n as the hydro­ c h l o r i d e s a l t i n female dogs weighing between 10 and 29 kg ( 2 2 ) . Q

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

10.

SATziNGER ET AL.

Etozolin: A Novel Diuretic

165

The doses administered ί.ν· ranged from 6 to 100 mg/kg. The animals were anesthetized by means og an i . v . i n j e c t i o n of 30 mg/kg p e n t o b a r b i t a l sodium (Nembutal ). In ,order to f i l l the e x t r a c e l l u l a r space with a s u f f i c i e n t f l u i d , 25 ml/kg i s o t o n i c s a l i n e s o l u t i o n was i n f u s e d i . v . w i t h i n one hour followed by continuous i . v . a d m i n i s t r a t i o n of f l u i d at a r a t e of 2 ml/min to which enough c r e a t i n i n e and PAH were added to achieve and maintain constant plasma l e v e l s of about 20 mg ? and 1.5 mg %, r e s p e c t i v e l y . A f t e r a one-hour p e r i o d of e q u i l i b r a t i o n , the u r i n e was c o l l e c t e d during 10 to 20 min periods by means of a c a t h e t e r . In the middle of each p e r i o d , blood was withdrawn from the femoral a r t e r y . Na , Κ and C l " as w e l l as c r e a t i n i n e and PAH were measured i n blood and u r i n e . Furthermore, the u r i n a r y output and the u r i n e pH were recorded. The f i r s t d i u r e t i c e f f e c t s were noted at about 20 mg/kg and reached almost maximal values a t 50 mg/kg a f t e r i . v . administratio e t o z o l i n reached i t s peak w i t h i n 1 hour a f t e r i . v . i n j e c t i o n . As i n the other s t u d i e s , there was a s l i g h t decrease i n c r e a t i ­ nine clearance, which i s used as a measure of GFR i n dogs. PAH clearance r a t e s dropped to o n e - t h i r d or even l e s s i n each t r i a l and, thus, approached the c r e a t i n i n e clearance v a l u e s . This s i g n i f i e d that the drug i n t e r f e r e d with PAH s e c r e t i o n i n the proximal tubule, so that PAH clearance could not be used to measure r e n a l plasma flow f o l l o w i n g a d m i n i s t r a t i o n of t h i s agent. The maximum tubular t r a n s p o r t c a p a c i t y f o r PAH (Ti°p^ ) was determined i n four separate s t u d i e s . A f t e r i . v . i n j e c t i o n of 50 mg/kg of e t o z o l i n , the mean Tmp value f e l l from 7-28 to 1.76 mg/min. Both the u r i n a r y e l e c t r o l y t e e x c r e t i o n and t o t a l u r i n a r y output increased by m u l t i p l e s of t h e i r o r i g i n a l values f o l l o w i n g a d m i n i s t r a t i o n of 50 mg/kg of e t o z o l i n and c h l o r i d e e x c r e t i o n c o n s i s t e n t l y exceeded that o f sodium. The most r e l i a b l e i n d i c a t o r of the potency of a d i u r e t i c , independent of body weight, i s i t s percentage of i n h i b i t i o n of e l e c t r o l y t e reabsorption. In the example i l l u s t r a t e d i n Table VI, sodium reabsorption f e l l to 83% and c h l o r i d e r e a b s o r p t i o n dropped to 80%. Tubular potassium reabsorption a l s o decreased, although no e x c r e t i o n l e v e l s were measured which were more than twice the b a s e l i n e values. Urine pH was lower i n a l l the t e s t s than i n the c o n t r o l p e r i o d s , i n d i c a t i n g an i n c r e a s e i n hydrogen i o n secretion. H

A{J

The next s t u d i e s were concerned with experimental changes i n pH. A l k a l o s i s was induced by i . v . i n f u s i o n of an NaHC0~ s o l u ­ t i o n and a c i d o s i s by i n f u s i o n of 0.1 Ν HC1. The a c t i o n of e t o z o l i n was s e v e r e l y i n h i b i t e d by metabolic a l k a l o s i s but was e s s e n t i a l l y unaffected by a c i d o s i s . In a s e r i e s of 18 t e s t s i n v o l v i n g 8 female dogs weighing 15 to 30 kg, a study was conducted to determine whether e t o z o l i n p o t e n t i a t e s the e f f e c t o f c h l o r o t h i a z i d e and/or chlormerodrin, or conversely, whether peak c h l o r o t h i a z i d e or chlormerodrin .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

creat) +

6.71 6.70 6.82 6.87 6.73 6.67 6.59

87.3 80.4 85.0 83.0 87.7 89.2 87.4

32.1 3.5 20.4 7.1 26.6 36.5 23.9

88.1 83.0 86.9 86.5 89.6 89.3 89.5

853 1233 1102 929 794 653 625

108 136 129 117 105 94 89

810 1088 981 793 684 554 550

75 69 64 57 55 52 54

48

45

52

40

46

42

36

5.4

8.0

7.6

6.5

5.8

4.7

4.7

30-50

50-70

70-90

90-110

110-130

130-150

50 mg/kg E t o z o l i n

10-30

7.27

65

292

221

59

96.5

Urine

67.0

Cl~

96.5

J

285

+

7.30

%

K

95.6

J

67.1

+

95.6

Na

420

οΓ

2.1

micro Eq/min

K

pH

-10- 0

+

Fractional Reabsorption

72

Na

U r i n a ry E x c r e t i o n

435

PAH

269

C

68

ml/min

v

GFR

3.0

Urine flow

(50 mg/kg) i . v . on Renal Function o f a Female Dog o f 17 kg

-20-10

min

Time

Effect of Etozolin

TABLE VI

10.

SATZINGER ET

AL.

Etozolin: A Novel Diuretic

167

d i u r e s i s can be enhanced by t h i s agent (23). E t o z o l i n was found to have an a d d i t i v e e f f e c t on the maximum Na , C l " and Κ e x c r e t i o n and u r i n a r y output produced by c h l o r o t h i a z i d e and by chlormerodrin. Likewise, c h l o r o t h i a z i d e and chlormerodrin enhanced the d i u r e s i s produced by e t o z o l i n . With the t e s t methods used, i t was not p o s s i b l e t o e s t a b l i s h whether these r e s u l t s were due to the three d i u r e t i c s having d i f f e r e n t mecha­ nisms of a c t i o n or d i f f e r e n t s i t e s o f a c t i o n i n the nephron. Micropuncture S t u d i e s i n Rats - The object o f performing d i u r e t i c s t u d i e s i n r a t s was to i n v e s t i g a t e the e f f e c t o f e t o z o l i n i n another s p e c i e s ( 2 2 , 2 4 J . F i r s t , dose-response curves were generated and then attempts were made to l o c a t e the s i t e of a c t i o n i n the nephron u s i n g micropuncture techniques (25). F i g u r e 3 shows the e f f e c t of i . g . a d m i n i s t r a t i o n o f the drug i n comparison to furosemid th four-hou u r i n a r output and Na , C l " and Κ e l i m i n a t i o n body weight. Etozolin y 1% tragacanth suspensions which were d i l u t e d with 0.2% saline s o l u t i o n . The c o n t r o l s r e c e i v e d the same volume o f tragacanth but without the d i u r e t i c . A l l the animals were then adminis­ tered 40 ml/kg of a 0.2% s a l i n e s o l u t i o n i . g . , so that the t o t a l volume o f f l u i d intubated equaled 5% o f the body weight. The t e s t groups c o n s i s t e d of 8 to 12 animals which were kept i n separate d i u r e s i s cages. Both e t o z o l i n and furosemide had d o s e - r e l a t e d d i u r e t i c e f f e c t s , furosemide being obviously the more potent of the two. Each compound produced a g r e a t e r i n c r e a s e i n c h l o r i d e e x c r e t i o n than i n sodium e x c r e t i o n . T h i s tendency seemed to be somewhat more pronounced f o r e t o z o l i n than f o r furosemide. Micropuncture Data - On the b a s i s of the dose-response curves, an i . v . dose o f 50 mg/kg of e t o z o l i n was s e l e c t e d f o r the micropuncture s t u d i e s . Male a n e s t h e t i z e d Sprague-Dawley r a t s were used. The animals were prepared f o r micropuncture i n the u s u a l way (25). Two to three l a t e proximal and e a r l y d i s t a l punctures were made, and the drug was i n j e c t e d i . v . A f t e r an i n t e r v a l o f 30 min, two to three more l a t e proximal and e a r l y d i s t a l t u b u l a r segments were micropunctured. The t u b u l a r f l u i d was c o l l e c t e d q u a n t i t a t i v e l y and the i n u l i n , sodium and potas­ sium c o n c e n t r a t i o n s were determined. Table VII shows the micropuncture data f o r the l a t e proximal nephron segments. T r a n s i t time was found to be extended a f t e r the a d m i n i s t r a t i o n of e t o z o l i n . None o f the other parameters measured i n the proximal convoluted tubule was s i g n i f i c a n t l y a f f e c t e d . Table VIII c o n t a i n s the micropuncture data o f the e a r l y d i s t a l nephron segments· E t o z o l i n caused a pronounced i n c r e a s e i n the flow r a t e o f t u b u l a r f l u i d i n the e a r l y d i s t a l t u b u l a r segments, a decrease i n the T F / P - i n u l i n r a t i o , an i n c r e a s e i n sodium and potassium c o n c e n t r a t i o n and a r i s e i n the TF/P-sodium and TF/P-potassium r a t i o s . F l u i d and e l e c t r o l y t e r e a b s o r p t i o n i n the loop o f Henle

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

O

Etozolin

Δ

Furoaemide

16

32

64

128

512

256

mg/kg

Figure 3. Comparison of the dose—response curves of etozolin and furosemide after i.g. administration to rats. Urinary output and Na* (top), CÏ and K fright) excretion over a four-hour period are shown (χ ± s , η = 8). The dotted lines represent the control values (χ ± s , η = 32). +

x

x

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

SATZINGER ET AL.

Etozolin: A Novel Diuretic

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

170

AGENTS

TABLE V I I Summary of the micropuncture data obtained by measure­ ment at the end of the proximal convoluted tubules o f s u p e r f i c i a l nephrons. T r a n s i t time i s the time from the d i f f u s e c o l o r a t i o n o f the kidney surface a f t e r l i s s a m i n e green i n j e c t i o n u n t i l the dye columns converge i n t o the t e r m i n a l p o r t i o n s o f the proximal convolution.

Control T r a n s i t Time (sec)

50 mg/kg i . v . s t a t ; then, 50 mg/kg/h i . v .

7.53+0.37 η = 6/12

10.8+0.86* η = 6/11

Nephron F i l t r a t i o n Rate (nl/min)

27.9+3.6 η = 7/11

22.3+2.7 η = 6/10

Flow Rate (nl/min)

10.3 + 0.7 η = 8/12

9.6 + 0.9 η = 7/12

TF P~

2.68 + 0.27 η = 7/12

TF Ρ TF Ρ

. i

n

u

Ί

.

l

i

+

+

n

2.49 + 0.37 η = 6/10

1.02 + 0.02 η = 7/12

0.99 + 0.02 η = 8/11

1.04 + 0.05 η = 7/12

0.97 + 0.04 η = 8/11

Mean + S.Ε.Μ.; η = number of animals/number of measurements; TF TF + — I n u l i n = tubular f l u i d to plasma i n u l i n r a t i o ; — Na = TF + tubular f l u i d to plasma sodium r a t i o ; —

Κ

= tubular

fluid

to plasma potassium r a t i o ; n l = n a n o l i t e r s ; *p <0.01.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

10.

SATZINGER E T A L .

171

Etozolin: A Novel Diuretic

TABLE V I I I Summary of the Micropuncture Data Obtained by Measurement at the E a r l y D i s t a l Tubular S i t e

Etozolin 50 mg/k i . v .

Nephron F i l t r a t i o n Rate (nl/min)

stat

25.2 + 4.4 η = 5/9

28.0 + 2.9 η = 6/11

Flow Rate (nl/min)

6.0 + 0.9 η = 6/11

8.9 + 1.0* η = 5/9

TF —

4.98 + 0.73 η = 6/11

2.72 + 0.30* η = 5/9

Inulin

E a r l y D i s t a l Sodium Concentration (mEq/L) E a r l y D i s t a l Potassium Concentration (mEq/L) T

F

— TF Ρ

XT

+

Na + K

78.5 + 8.5** η = 5/9

42.4 + 3.5 η = 6/11

1.12 + 0.09 η = 6/11

2.52 + 0.41** η = 5/9

0.274 + 0.037 η = 6/11

0.543 + 0.048** η = 5/9

0.270 + 0.031 η = 6/11

0.615 + 0.074** η = 5/9

Mean + S.Ε.Μ.; η = number of animals/number of measurements; TF TF + — I n u l i n = t u b u l a r f l u i d to plasma i n u l i n r a t i o ; — Na = TF + t u b u l a r f l u i d to plasma sodium r a t i o ; —

Κ

= tubular

fluid

to plasma potassium r a t i o ; *p <0.01; n l = n a n o l i t e r s ; **p <0.001.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

172

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can be c a l c u l a t e d from the l a t e proximal and e a r l y d i s t a l data. Under the i n f l u e n c e of the drug, f l u i d r e a b s o r p t i o n i n t h i s nephron segment f e l l from 41.7 to 7·3Ϊ, sodium r e a b s o r p t i o n from 82.9 to 48.7% and potassium reabsorption-from 83.3 to 36?. It can be concluded from these f i n d i n g s that d i u r e t i c and n a t r i ­ u r e t i c a c t i o n of e t o z o l i n d e r i v e s from the substance's i n h i b i ­ t i o n of f l u i d and e l e c t r o l y t e r e a b s o r p t i o n i n the loop of Henle. The major metabolite of e t o z o l i n , (Z)-ZT-methyl-4-oxo-5-(lp i p e r i d i n y l ) - 2 - t h i a z o l i d i n y l i d e n J 7 a c e t i c a c i d , was a l s o subjected to d e t a i l e d pharmacological i n v e s t i g a t i o n . I t possesses potent d i u r e t i c p r o p e r t i e s and i t s s i t e of a c t i o n i s a l s o located i n the ascending limb of Henle*s loop. A l l the r e s u l t s suggest that t h i s metabolite plays a major part i n the b i o l o g i c a l a c t i v i t y observed f o r e t o z o l i n . General Pharmacology - Extensive pharmacological screening did not r e v e a l any r e s u l t d i u r e t i c a c t i o n of e t o z o l i n substance to known a c t i v e c h o l e r e t i c compounds prompted us to undertake s t u d i e s to compare e t o z o l i n and p i p r o z o l i n (Compound 13) i n r a t s . P i p r o z o l i n was a l s o evaluated f o r d i u r e t i c ac­ t i v i t y i n r a t s . Conscious animals weighing 160 to 200 g were employed. They were administered the t e s t substance together with 5 ml/100 g body weight of a 0.2% s a l i n e s o l u t i o n and the u r i n e was c o l l e c t e d over a 4 hour p e r i o d . Rats weighing 200 to 250 g, anesthetized with p e n t o b a r b i t a l were used f o r the chol e r e s i s t e s t s . The e f f l u e n t b i l e was measured f o r 120 minutes with a drop counter. The r e s u l t s of these s t u d i e s are presented i n F i g u r e s 4 and 5. Despite the c l o s e chemical r e l a t i o n s h i p of the two com­ pounds, e t o z o l i n showed only a s l i g h t c h o l e r e t i c a c t i o n compared to p i p r o z o l i n . While 100 mg/kg of p i p r o z o l i n caused a r a p i d i n c r e a s e i n b i l e s e c r e t i o n l a s t i n g over 90 minutes, only a s l i g h t , slow i n c r e a s e i n b i l e s e c r e t i o n was observed a f t e r e t o z o l i n . Even 250 mg/kg of e t o z o l i n produced a marginal i n ­ crease which subsided a f t e r 60 minutes, while 200 mg/kg of p i p r o z o l i n had a stronger d o s e - r e l a t e d e f f e c t than d i d 100 mg/kg. While 50, 100 and 200 mg/kg of e t o z o l i n administered i n t r a g a s t r i c a l l y caused a dose-related i n c r e a s e i n u r i n e e l i m i ­ n a t i o n , p i p r o z o l i n showed no s i g n i f i c a n t e f f e c t at the same doses. An important f a c t o r i n long-term treatment with d i u r e t i c s i s t h e i r e f f e c t on glucose metabolism. Therefore, corresponding glucose tolerance t e s t s were conducted i n connection with the long-term experiments i n r a t s and dogs (26). In r a t s , the dosages were 50, 250 and 2,000 mg/kg/day; the d u r a t i o n of the experiment was 18 months. Oral doses o f 30, 120 and 480 mg/kg/day were administered to dogs f o r 12 months. A glucose t o l e r ­ ance t e s t was conducted i n dogs a f t e r the 1st, 4th, 8th, 12th, 26th, 39th and 52nd week and i n r a t s a f t e r the 26th and 78th week. Two g of glucose/kg of body weight were administered as a

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

n=6

n=6

n=6

n=6

ki 30- 60

H 60-90

20H

40·

J

n=7

0-30

η =11

j

250 mg/kg i.d.

n=7

30-60

60-90

I

Ύ

1

n=7

n=5

j Piprozolin 200 mg/kg i.d.

ΙΧΛ E t o z o l i n

n=4

90-120 mi η

η =5

Figure 4. Effect of etozolin (100 and 250 mg/kg) and piprozolin (100 and 200 mg/kg) on bile secre­ tion in anesthetized rats following i.d. administration

α 20Η

n=7

60

100

60Η

n= 7

100 mg/kg i.d.

j Piprozolin

Etozolin

80H

J

kVI

80Η

ΙΟΟΊ

«π. Ο Ν Ο

•

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20% s o l u t i o n v i a a stomach tube. F o l l o w i n g an 18 hour food abstinence p e r i o d , serum glucose was measured before administ r a t i o n and then 1 hour and 4 hours a f t e r a d m i n i s t r a t i o n o f the drug. There were no d i f f e r e n c e s between the animals t r e a t e d and the c o n t r o l s a t any time during the experiment. Thus, the p o s s i b i l i t y o f glucose i n t o l e r a n c e i n r a t s and dogs can be excluded even when high doses o f e t o z o l i n are given over long p e r i o d s o f time. A n t i h y p e r t e n s i v e E f f e c t s i n Rats - The a n t i h y p e r t e n s i v e p r o p e r t i e s o f e t o z o l i n were t e s t e d i n conscious r a t s with hypertension o f d i f f e r e n t o r i g i n : (a) induced by desoxyc o r t i c o s t e r o n e acetate (DOCA), (b) h e r e d i t a r y and (c) renov a s c u l a r hypertension caused by experimental impairment o f the r e n a l c i r c u l a t i o n . Blood pressure was measured n o n - i n v a s i v e l y at the base o f the t a i l e i t h e r v i a a condenser microphone or plethysmographically. E t o z o l i g a s t r i c a l l y suspended i day. The r e s u l t s o f these t e s t s are shown i n F i g u r e s 6, 7 and 8. As we had expected, the most pronounced e f f e c t s were observed i n the DOCA-treated r a t s , s i n c e the impaired sodium balance was r e s t o r e d to i t s normal l e v e l and the volume o f e x t r a c e l l u l a r f l u i d was reduced as a r e s u l t o f the d i u r e t i c a c t i o n of e t o z o l i n . The blood pressure was a l s o s i g n i f i c a n t l y lowered i n r a t s with renovascular hypertension and i n animals with spontaneous hypertension. Since the three types o f hypert e n s i o n are caused by d i f f e r e n t mechanisms, there i s reason to b e l i e v e that the a n t i h y p e r t e n s i v e e f f e c t o f e t o z o l i n d e r i v e s not only from a r e d u c t i o n o f the plasma volume, but a l s o from p e r i p h e r a l mechanisms, p o s s i b l y of the same kind as are postul a t e d f o r the t h i a z i d e s . METABOLISM AND

PHARMACOKINETICS

The f a t e o f e t o z o l i n i n the organism was i n v e s t i g a t e d u s i n g C-etozoliri l a b e l e d i n the 2 - p o s i t i o n o f the t h i a z o l i d i n e r i n g (27). The absorption o f C - e t o z o l i n was > 90% i n man, > 80% i n £|ts and to a l e s s e r degree i n dogs. In a l l s p e c i e s , maximum C blood l e v e l s were observed 2 to 3 hours a f t e r o r a l administ r a t i o n . A f t e r 24 hours, the c o n c e n t r a t i o n o f r a d i o a c t i v e m a t e r i a l i n the blood had dropped to approximately 8% of the maximum values i n r a t s and dogs and to approximately 15% i n man. In r a t s , the blood l e v e l s can be d e s c r i b e d by a one-compartment body model, the absorption h a l f - l i f e being approximately 0.6 hour, and the e l i m i n a t i o n h a l f - l i f e being approximately 6 hours. During absorption, e t o z o l i n underwent a d i s t i n c t f i r s t - p a s s effect. In a l l s p e c i e s , the majority o f the plasma r a d i o a c t i v i t y corresponded to metabolite I (see F i g u r e 9), which a l s o has d i u r e t i c a c t i v i t y . Unchanged drug appeared i n plasma i n c o n s i d e r a b l y lower c o n c e n t r a t i o n s . In r a t s , more than 90% o f plasma r a d i o a c t i v i t y could be a s c r i b e d to metabolite I. In man,

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

SATZINGER ET A L .

Etozolin: A Novel Diuretic

|··ν|

Control Etozolin

j

Figure 5.

I Piprozolin

Effect of increasing doses of etozolin and piprozolin on urine excretion in conscious rats

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

175

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Ω

I

Figure 7. Effect of repeated i.g. treatment with 150 mg/kg of etozolin on the blood pressure of rats with spontaneous hypertension + = ρ > 0.05 g

-α

10.

SATZINGER ET AL.

Etozolin: A Novel Diuretic

177

Figure 8. Effect of etozolin on systolic and diastolic blood pressure in rats with renovascular hypertension. Experimental and control groups consisted of 10 animals each.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 9. Proposed metabolic pathway of etozolin in rats, dogs, and man

Ω

Η·»

10.

sATziNGER ET AL.

Etozolin: A Novel Diuretic

179

approximately 80% corresponded to the sum of metabolite I and the unchanged drug during the 0 to 8 hour observation p e r i o d . In a l l s p e c i e s , the main p o r t i o n o f the blood r a d i o a c t i v i t y was present i n the plasma. In v i t r o i n v e s t i g a t i o n o f human plasma r e v e a l e d a low p r o t e i n b i n d i n g f o r e t o z o l i n and metabolite I (approximately 45% and 35%, r e s p e c t i v e l y ) . In r a t s , approximately 80% o f the r a d i o a c t i v i t y was e l i m i ­ nated i n the u r i n e and the h a l f - l i f e was 6 hours; i n man ap­ proximately 90% was e l i m i n a t e d i n the u r i n e and the h a l f - l i f e was 8.5 hours. The r e n a l C e l i m i n a t i o n i n dogs ranged between 29% and 72%. In man, almost 100% o f the r a d i o a c t i v e dose was r e c o v e r e d i n the excrement w i t h i n four days. In r a t s , the highest C c o n c e n t r a t i o n s were found i n blood, kidneys and liver. In the other organs, the concentrations were g e n e r a l l y much lower, the lowest bein i th b r a i (27.) Autoradiograph ojj r a t s f o l l o w i n g repeate C-etozolin/kg/day fro y pregnancy s u b s t a n t i a l l y lower r a d i o a c t i v i t y c o n c e n t r a t i o n s i n the f e t u s e s than i n the mothers (28). The r a d i o a c t i v i t y was d i s t r i b u t e d uniformly throughout the f e t a l organs, the lowest c o n c e n t r a t i o n s o c c u r r i n g i n the b r a i n . The p l a c e n t a would appear to a c t as a b a r r i e r , r e s t r i c t i n g the passage o f r a d i o a c t i v e compounds from the mother's c i r c u l a t i o n i n t o the f e t a l t i s s u e . The s t r u c t u r e s o f the metabolites were e l u c i d a t e d i n s t u d i e s u s i n g r a t , dog and Imuran u r i n e and r a t b i l e obtained a f t e r o r a l a d m i n i s t r a t i o n o f C - e t o z o l i n (29). Seven metabolites were i s o l a t e d . The metabolic pathway o f e t o z o l i n i s the same i n r a t s , dogs and man, and i s c h a r a c t e r i z e d i n three steps (Figure 9 ) : 1) enzymatic cleavage of the e s t e r group, which leads to the main metabolite (metabolite I) i n the plasma of a l l 3 s p e c i e s , 2) g l u c u r o n i d a t i o n of the r e s u l t i n g metabolite I, l e a d i n g to metabolites I I and I I I , which are d i a s t e r e o i s o m e r i c e s t e r s o f the two enan­ t i o m e r i c forms o f metabolite I with β-D-glucuronic a c i d and 3) o x i d a t i o n of the p i p e r i d i n e moiety to metabolites IV-VII. In a l l s p e c i e s , approximately 50-60% o f the u r i n a r y r a d i o ­ a c t i v i t y corresponded to the sum o f the f r e e metabolite I and the metabolite I g l u c u r o n i d e s . The unconjugated form was more abundant i n r a t s , while the metabolite I glucuronides predomi­ nated i n man. Intermediate r e s u l t s were obtained using dogs. No unchanged substance was found i n the u r i n e of any o f the s p e c i e s i n v e s t i g a t e d . There was l i t t l e variance i n the excre­ t i o n r a t i o s and u r i n a r y metabolite p r o f i l e s i n r a t s f o l l o w i n g the o r a l i n t u b a t i o n of d i f f e r e n t doses (3-100 mg/kg) o f the drug, i n d i c a t i n g that e t o z o l i n s metabolism i s not dose r e l a t e d i n t h i s range. A s p e c i f i c TLC a n a l y t i c a l method was used to study the pharmacokinetics of the unchanged substance and i t s p r i n c i p a l metabolite (metabolite I) f o l l o w i n g s i n g l e and repeated adminis­ t r a t i o n o f v a r i o u s doses by d i f f e r e n t routes (30). The pharma­ c o k i n e t i c behavior o f e t o z o l i n and metabolite I could be 1

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

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d e s c r i b e d by f i r s t - o r d e r process models i n each study. Etozolin was metabolized f a i r l y r a p i d l y i n each o f the s p e c i e s i n v e s t i gated, only r e l a t i v e l y low c o n c e n t r a t i o n s being found i n plasma and none i n u r i n e . No deep compartment d i s t r i b u t i o n (e.g., p r o t e i n binding) was detected. Minor d i f f e r e n c e s were observed i n the e x c r e t i o n o f the p r i n c i p a l metabolite ( h a l f - l i f e 4-8 hours), but the uniform d i s t r i b u t i o n volume o f approximately 1 L/kg i n d i c a t e s that the m e t a b o l i t e s apparent volume o f d i s t r i bution i s i d e n t i c a l i n a l l three s p e c i e s . Each s p e c i e s was administered a d i f f e r e n t dose range. The pharmacokinetic behavior o f e t o z o l i n and i t s p r i n c i p a l metabol i t e was not dose r e l a t e d i n the ranges i n v e s t i g a t e d (5-50 mg/kg i . v . i n r a t s , 30-480 mg/kg i . g . i n dogs, 400-800 mg o r a l l y i n man). The plasma l e v e l time-curves f o r e t o z o l i n and metabolite I i n man are shown i n F i g u r e 10 E t o z o l i n was i n v e s t i g a t e dogs. The human study f u n c t i o n . The pharmacokinetics o f e t o z o l i n and metabolite I were unaffected by the s i z e o f the m u l t i p l e doses g i v e n . I t has been demonstrated that n e i t h e r the parent substance nor metabol i t e I i s l i k e l y to accumulate i n the organism i f the recommended t h e r a p e u t i c dosage i n t e r v a l s are observed. 1

CLINICAL STUDIES Comparative S t u d i e s o f the D i u r e t i c A c t i v i t y i n Normal Volunteers - The d i u r e t i c e f f e c t o f e t o z o l i n was f i r s t i n v e s t i gated i n 8 healthy volunteers (J31). A c l e a r dose-dependent d i u r e t i c e f f e c t could be demonstrated. When compared to mefrus i d e as a r e f e r e n c e substance, both compounds showed a s i m i l a r d i u r e t i c p r o f i l e . The maximum e f f e c t occurred 2 to 4 hours a f t e r o r a l a d m i n i s t r a t i o n ; the d u r a t i o n o f the i n c r e a s e d d i u r e s i s being about 10 hours. However, a t the doses used, the t o t a l volume excreted was s i g n i f i c a n t l y higher with e t o z o l i n than with mefruside ( F i g u r e 11). The d i u r e t i c p r o f i l e o f e t o z o l i n i s d i f f e r e n t from that o f furosemide, as shown by the t o t a l u r i n a r y output a f t e r e q u i e f f e c t i v e doses. Furosemide, i n c o n t r a s t t o e t o z o l i n and the t h i a z i d e d i u r e t i c s , has a very r a p i d onset o f d i u r e s i s , l a s t i n g about 2 hours with a r a p i d decrease i n u r i n e output followed by a d i s t i n c t rebound phenomenon l a s t i n g over 12 hours ( F i g u r e s 12, 13, 14). A n t i h y p e r t e n s i v e A c t i v i t y - In a c o n t r o l l e d t r i a l i n pat i e n t s with e s s e n t i a l hypertension, 200 mg of e t o z o l i n or h a l f the d i u r e t i c dose showed an a n t i h y p e r t e n s i v e e f f e c t s i m i l a r t o that o f a drug combination c o n s i s t i n g o f 150.0 mg o f i n o s i t o l n i c o t i n a t e + 15 mg mefruside + 0.15 mg r e s e r p i n e ( F i g u r e 15). In another c o n t r o l l e d study (32), l a s t i n g over 6 months, a pronounced a n t i h y p e r t e n s i v e e f f e c t could be observed ( F i g u r e 16).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 10. Plasma level time curves of the unchanged drug (top right) and metabolite I (bottom) after oral administration of 800 mg of etozolin to man. The 95% confidence interval of the function and the measured plasma level concentrations are also shown. oo

DIURETIC

182

Figure 11. η = 8; (

Mean value of urine volume. ( ) Etozolin, 800 mg, ) mefruside, 50 mg, η = 8; (+ +) placebo, η = 8.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

I

17.00

l

20.00

l

Figure 12.

23.00

ι

ι

13.00

ι

17.00

ι

20.00

ι

23.00

Dose and drug*dependent urinary excretion of sodium

9.00

Ω

Figure 13. Dose and drug-dependent urinary excretion of potassium ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

00

I

I

23.00

mval/h

20.00

Δ Cl

Figure 14.

13.00

17.00

ι

20.00

I

23.00

Dose and drug-dependent urinary excretion of chloride

9.00

Furosemid 80 mg

Furosemid 40 mg

E t o z o l i n 800 mg

E t o z o l i n 400 mg

8

I

Ο

1*1

3

©

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 15. Curves of mean blood pressure values in a sitting position during six months therapy with etozolin and with a reference substance (combination)

Ο

g

h-*

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

•

Figure 16.

1 2. month

1 3. month

Therapy with Etozolin and Reference substance —

4.wk.

«

1 4. month

1

~~

5. month

T~

^

) and with

6. month

Mean blood pressure values in a sitting position during six months therapy with etozolin ( a reference substance (combination) ( )

<

Duration of therapy

3.wk.

r-f-.

1.wk.2.

188

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Side E f f e c t s - Out o f 447 p a t i e n t s with edema o f v a r y i n g e t i o l o g y , 29 (6.49$) reported side e f f e c t s : nausea, 2.0%; vomiting, 1.6$; d i z z i n e s s , O.2$; headache, O.7$; g a s t r o i n t e s ­ t i n a l complaints, O.9$; s k i n rashes, O.9$· Some p a t i e n t s complained o f two o r more s i d e e f f e c t s . I t i s y e t t o be e s t a b l i s h e d whether these s i d e e f f e c t s are a t t r i b u t a b l e t o e t o z o l i n alone s i n c e other drugs were administered concomi­ t a n t l y . The c h a r a c t e r i s t i c side e f f e c t s o f d i u r e t i c and/or hypotensive therapy, i . e . , hyperuricemia and e l e v a t i o n o f c r e a t i n i n e l e v e l s a l s o occurred with e t o z o l i n .

Literature Cited 1. Clodi, P. H. and Schnack, Η., Wiener k l i n . Wschr., (1966), 78, 774. 2. Campese, V. M. and Siro-Brigiani, G . , B o l l . Soc. I t a l . b i o l . sperim., (1971) 3. Maxwell, D. R., Szwed and K l e i t , S. Α . , Am. J. Physiol., (1974), 226, 540. 4. Heintze, K . , Gotz, R. and Koerlings, Η . , NaunynSchmiedeberg's Arch. Pharm., (1977), 297 (Suppl. 2), 150. 5. Satzinger, G . , Arzneim.-Forsch./Drug Research, (1977), 27, (9a), 1742. 6. Greven, J. and Heidenreich, O., Arzneim.-Forsch./Drug Research, (1977), 27, (9a), 1755. 7. Burg, Μ., Stoner, L., Cardinal, J. and Green, Ν . , Am. J. Physiol., (1973), 225, 119. 8. Burg, M. and Green, Ν . , Kidney I n t . , (1973), 4, 301. 9. Jacobson, H. R. and Kokko, J. Η . , Ann. Rev. Pharm., (1976), 16, 201. 10. Ebel, H . , Naunyn-Schmiedeberg's Arch. Pharm., (1974), 281, 301. 11. Meng, K. and Loew, D . , "Diuretika", pp. 3,7,8,11, Georg Thieme, Stuttgart, 1974. 12. Price, C. C. and Oae, S., "Sulfur Bonding", p. 61, Ronald, Ν. Υ . , 1962. 13. Dupont, L . and Dideberg, O., Acta Cryst., (1972), B28, 2340. 14. Yukawa, Y., "Handbook of Organic Structural Analysis", pp. 518, 519, W. A. Benjamin Inc., Ν. Y., 1965. 15. Harshbarger, W. R. and Bauer, S. Η . , Acta Cryst., (1970), B26, 1010. 16. Higgs, M. A. and Sass, R. L., Acta Cryst., (1963), 16, 657. 17. Pauling, L., "Die Natur der chemischen Bindung", p. 108, Verlag Chemie, Weinheim/Bergstrasse, 1962. 18. M i l l e r , R. A. L., Robertson, J. Μ., Sim, G. Α . , Clapp, R. C., Long, L . and Hasselstrom, T . , Nature, (1964), 202, 287. 19. Gunther, T. and Ahlers, J., Arzneim. Forsch./Drug Research, (1976), 26, (1), 13.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

10.

SATZINGER ET AL. Etozolin: A Novel Diuretic

189

20. Schultz, E . M . , Smith, R. L . and Woltersdorf, O. W., Jr., Ann. Rep. Med. Chem., (1975), 10, 71. 21. Weber, Ε . , "Grundri der biologischen Statistik", p. 582, Gustav Fischer Verlag, Jena, 1972. 22. Heidenreich, O., Gharemani, G . , Keller, P . , Kook, Y. and Schmiz, K . , Arzneim.-Forsch./Drug Research, (1964), 14, 1242. 23. Heidenreich, O. and Baumeister, L., K l i n . Wschr., (1964), 42, 1236. 24. Herrmann, M . , Bahrmann, H . , Birkenmayer, E., Ganser, V . , Heldt, W. and Steinbrecher, W., Arzneim.-Forsch./Drug Research, (1977), 27, 1745. 25. Greven, J. and Heidenreich, O., Arzneim.-Forsch./Drug Research, (1977), 27, 1755. 26. Herrmann, Μ., Wiegleb, J. and Leuschner, F., Arzneim.Forsch./Drug Research (1977) 27 1758 27. Vollmer, K . - O . , v V. and Hengy, Η . , Arzneim.-Forsch./Drug , (1977), , 1767. 28. Franklin, E . R., Chasseaud, L . F . and Taylor, T . , Arzneim. Forsch/Drug Research, (1977), 27, 1800. 29. V. Hodenberg, Α . , Vollmer, K . - O . , Klemisch, W. and Liedtke, B . , Arzneim.-Forsch./Drug Research, (1977), 27, 1776. 30. Gladigau, V. and Vollmer, K . - O . , Arzneim.-Forsch./Drug Research, (1977), 27, 1786. 31. Biamino, G . , Arzneim.-Forsch./Drug Research, (1977), 27, 1814. 32. Felix, G . , Windsheimer, F . and Otrzonsek, G . . Private Communication. RECEIVED August 21, 1978.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11 The Evolution of the (Aryloxy)acetic Acid Diuretics O. W. WOLTERSDORF, JR., S. J. D E SOLMS, and E . J. CRAGOE, JR. Merck Sharp and Dohme Research Laboratories, West Point, PA 19486

The advent of moder discovery of the diureti spite of the tremendous advances in the f i e l d , the mercurial diuretics, particularly the phenoxyacetic acids, merbaphen (Compound 1, Figure 1) and mersalyl (Compound 2, Figure 1) possess many pharmacodynamic attributes which are as good as, or superior to, the modern agents, including potent saluresis, proper urinary Na /Cl balance, an acceptable potassium excre­ tion profile and uricosuric a c t i v i t y . In addition, much is known concerning their mechanism of action. Cafruny(3) and others have shown that the mercurials react with sulfhydryl­ -containing compounds both in vitro and in vivo and that such a reaction at the renal receptor i s associated with the observed saluresis and diuresis. Also, the concentration of protein­ -bound sulfhydryl groups in renal cells is minimal when the concentration of mercury is maximal, which is also the time when diuresis is maximal. The advantages of the mercurials were more than outweighed by their disadvantages which included tachyphylaxis, poor oral efficacy and toxicity. Using the mercurial phenoxyacetic acids as models, a search was initiated in our laboratories by J. M. Sprague and co-workers for a non-mercurial agent which would lack the drawbacks of the mercurials but would possess their diuretic properties. Our approach to the solution of this problem was to search for a chemical moiety that would mimic the chemical behavior of the mercury atom in the diuretic mercurials, especially as i t pertains to reactions with sulfhydryl groups. After a long investigation, the a,β-unsaturated ketones (3) were found to react by a Michael-type reaction with standard sulfhydrylcontaining substances in a parallel fashion to mercurials (4) even under physiological conditions of pH and temperature as shown by the two equations in Figure 2. Acryloylbenzoic acids of the type 3 were known at the time, but when synthesized proved to be inactive as diuretics. This +

-

0-8412-0464-0/78/47-083-190$10.25/0 © American Chemical Society In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

(Aryloxy)acetic

WOLTERSDORF ET AL.

2

H-Hg-R

+

Acid

Diuretics

2

RSH ~ — ^ R-S-Hg~R

(4) Figure 2. Reaction of α,β-unsaturated ketones and mercurials with mercaptans

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

191

DIURETIC

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AGENTS

was d i s a p p o i n t i n g , but a knowledge of the c r i t i c a l s t r u c t u r a l requirements of the organic moiety f o r m e r c u r i a l s l e d us t o prepare an organic p o r t i o n of the α,β-unsaturated ketones more a k i n to that of merbaphen. This was achieved i n compound 4 which, to our d e l i g h t , had d i u r e t i c p r o p e r t i e s . The a c t i v i t y was very weak and o f short d u r a t i o n , but i t had the q u a l i t a t i v e p r o p e r t i e s we were seeking. Thus, a lead had been e s t a b l i s h e d which, upon development, u l t i m a t e l y l e d to the discovery o f e t h a c r y n i c a c i d (Compound 5, Figure 1) ( 4 ) .

(3) The information generated by the research i n the e t h a c r y n i c a c i d s e r i e s set o f f an e x p l o s i o n of ideas on the design of new d i u r e t i c s both i n our l a b o r a t o r i e s and i n o t h e r s . The h i s t o r y o f how t h i s family of d i u r e t i c s evolved i s i l l u s t r a t e d g r a p h i c a l l y i n Figure 1. The arrows portray the g e n e o l o g i c a l r e l a t i o n s h i p of each member of the family which w i l l be discussed system­ a t i c a l l y i n t h i s paper. I n t e r e s t i n g l y , many of these s t r u c t u r a l types e x h i b i t q u a l i t a t i v e l y and q u a n t i t a t i v e l y d i f f e r e n t r e n a l characteristics. (Acryloylphenoxy)acetic Acids - A f t e r having e s t a b l i s h e d com­ pound 4 as a lead, i t s development i n v o l v e d a systematic s t r u c ­ t u r a l v a r i a t i o n and c o r r e l a t i o n of b i o l o g i c a l a c t i v i t y with a s e r i e s of p h y s i c a l p r o p e r t i e s , i n c l u d i n g pKa, H 0 / l i p i d d i s t r i ­ b u t i o n , p r o t e i n binding, e t c . In a d d i t i o n , c o r r e l a t i o n of some chemical p r o p e r t i e s was i n v e s t i g a t e d . The compounds were i n i t i a l l y screened i n t r a v e n o u s l y i n dogs where the sodium, potassium and c h l o r i d e e x c r e t i o n and u r i n e volumes were measured i n comparison to c o n t r o l s . For convenience, the compounds were scored according to the c r i t e r i o n i n Table I . We f i r s t i n v e s t i g a t e d the optimum l o c a t i o n o f the oxyacetic a c i d group i n r e l a t i o n to the s u b s t i t u t e d a c r y l o y l moiety of compound 4 (Table I I ) . When these groups were ortho to each other, the compound was i n a c t i v e . With the meta-orientation, a c t i v i t y was improved, but the p a r a - o r i e n t a t i o n was optimal. Next, a t t e n t i o n was focused on the r o l e of nuclear subs t i t u e n t s (Table I I I ) . Only marginal a c t i v i t y was seen when the four nuclear s u b s t i t u e n t s were hydrogen, and s u b s t i t u e n t s i n the 2 - p o s i t i o n made l i t t l e c o n t r i b u t i o n to a c t i v i t y . In the 3p o s i t i o n , although f l u o r o had l i t t l e e f f e c t , the other halo, methyl and t r i f l u o r o m e t h y l s u b s t i t u e n t s bestowed a marked i n ­ crease on the a c t i v i t y . The combined e f f e c t o f two c h l o r o 2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

woLTERSDORF

ET AL.

(Aryloxy)acetic Acid Diuretics

TABLE I S c o r i n g System Dog Assay

5 mg/kg i . v . Stat Dose Score 0

0-99

+

A c t i v e Above 5 mg/kg

1

100 - 399

2

400 - 599

3

600 - 799

4

800 - 899

5

900 - 999

6

>1000

Female animals were starved overnight, anesthetized with phénobarbital, c r e a t i n i n e primed, c a t h e t e r i z e d and i n f u s e d with phosphate b u f f e r a t a r a t e of 3 ml/min. The drug was given i . v . a t 5 mg/kg over a p e r i o d o f 5 min, and 15-min c o l l e c t i o n s of u r i n e were taken over a p e r i o d of 2 h. The data recorded were the average of the two highest consecutive 15-min c o l l e c t i o n s .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

193

DIURETIC

194

TABLE I I E f f e c t o f L o c a t i o n of the A c r y l o y l Moiety 3

CH COOH 2

Ring P o s i t i o n of C H_—C

C—

0

2

5

II CH

II 2

CI

I.V. Dog N a Scores

+

—

0

2

4

+

3

4

2

4

3

4

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

11.

WOLTERSDORF ET AL.

195

(Aryloxy)acetic Acid Diuretics

TABLE I I I E f f e c t of Nuclear S u b s t i t u e n t s 3

0

CH COOH 2

- Substituents— 5 3

2 H

H

(MK-495) C l

H

H

H

H

H

CH

3

I . J . Dog Na Score

6

a

T l/2 T

+

27

H

0

>90 27

H

F

H

H

+

H

Cl

H

H

4

7

H

Br

H

H

4

9

H

I

H

H

4

9

H

CF

H

H

3

14

H

CH

3

H

H

3

48

H

C H

5

H

H

+

43

(MK-595) C l

Cl

H

H

6

6

Cl

H

Cl

H

+

6

H

Cl

Cl

H

3

6

Cl

H

H

Cl

+

22

H

H

5

45

H

0

2

(MK-695) CH

3

CH

3

CH

a

3

3

CH

3

CH

H H

CH

3

CH

CH

3

C H

CH

3

3

3 CH

3

3

H

CH

CH

H

3

H CH

3

3

58

0

165

+

75

CH

3

+

73

CH

3

+

60

Time i n minutes r e q u i r e d f o r 50% r e a c t i o n with a standard s o l u t i o n of mercaptoacetic a c i d .

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

196

(MK-595, e t h a c r y n i c a c i d ) or two methyl groups (MK-695) l o c a t e d i n the 2- and 3 - p o s i t i o n s produced a dramatic i n c r e a s e i n d i u r e t i c a c t i v i t y . Other o r i e n t a t i o n s of two c h l o r o or methyl groups were much l e s s e f f e c t i v e and each of the t r i m e t h y l and the tetramethyl analogs were v i r t u a l l y i n a c t i v e . As we had a n t i c i p a t e d , the ( a c r y l o y l p h e n o x y ) a c e t i c a c i d s and the m e r c u r i a l d i u r e t i c s e x h i b i t e d marked s i m i l a r i t i e s i n t h e i r r e a c t i o n with s u l f h y d r y l - c o n t a i n i n g compounds, both i n v i t r o and i n v i v o (3^, 4^ 5}. A measure of t h i s property was recorded f o r each of our compounds as i l l u s t r a t e d i n the column headed Τ,, which i s a c t u a l l y the time i n minutes r e q u i r e d f o r each compound to g i v e a 5 0 Ï complete r e a c t i o n with a standard c o n c e n t r a t i o n of mercaptoacetic a c i d at pH 7.4. These data i n d i c a t e that the most a c t i v e compounds r e a c t very r a p i d l y with t h i s model s u l f h y d r y l reagent Although as one might expect some compounds which r e a c d i u r e t i c a c t i v i t y , and a c t i v e , e.g., the f i r s t compound, because they f a i l to meet other s t r u c t u r a l or chemical requirements. Compounds c o n t a i n i n g 5- and 6 - s u b s t i t u e n t s were markedly l e s s a c t i v e . T h i s l e d us to b e l i e v e that e i t h e r the 2- and 3 - p o s i t i o n s (or the 5- and 6- p o s i t i o n s ) must remain u n s u b s t i t u t e d to maintain a c t i v i t y . As w i l l be seen l a t e r , t h i s i d e a was d r a m a t i c a l l y disproved i n two instances. A few of the compounds that were prepared to evaluate the e f f e c t of s u b s t i t u e n t s on the v i n y l carbon atoms appear i n Table IV. The f i r s t compound (where each R=H) r e a c t s so r a p i d l y and i r r e v e r s i b l y with many n u c l e o p h i l e s ( i n c o n t r a d i s t i n c t i o n to the other compounds i n t h i s s e r i e s ) that i t possesses very l i t j l e a c t i v i t y . Each of the compounds bearing a s i n g l e alpha (R ) s u b s t i t u e n t , whether a l k y l or c y c l o a l k y l , e x h i b i t e d potent d i u r e t i c a c t i v i t y and a r a p i d r e a c t i o n with mercaptoacetic a c i d . The detrimental e f f e c t of an a l k y l s u b s t i t u e n t on both the alpha (R ) and the beta (R ) carbon atom was even more pronounced when a l k y l groups occupied both beta (R and R ) atoms. Hydroxy and methoxy groups on the beta (R ) carbon atom were not t o l e r a t e d . S e v e r a l compounds were s t u d i e d i n which the ether oxygen atom was replaced by other f u n c t i o n a l i t i e s (Table V). Replacement of oxygen by s u l f u r provided compounds that were as potent as t h e i r oxygen i s o s t e r e s ; however, the corresponding s u l f o x i d e s possessed l i t t l e a c t i v i t y . Replacement of oxygen by -NH-, -N(CH^)- or -CH^- d r a s t i c a l l y reduced or a b o l i s h e d a c t i v i t y . However, replacement of the oxygen atom by a bond provided the corresponding p h e n y l a c e t i c a c i d s which possessed modest activity. Representatives of the many groups which were evaluated as surrogates f o r the carboxymethyl moiety (R) are l i s t e d i n Table VI. When a methylene proton i s replaced by f l u o r o , f u l l act i v i t y i s maintained. However, s u b s t i t u t i o n of one methylene proton by a methyl group i s d e t r i m e n t a l and s u b s t i t u t i o n of both 3

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

WOLTERSDORF ET AL.

(Aryloxy)acetic Acid Diuretics

197

TABLE IV E f f e c t of Substituents on the V i n y l Carbon Atoms

Ρ il

N

1

CH COOH 2

I.V. Dog R

1

R

2

R

3

Na

+

T

Score

l/2

-H

Η

Η

+

<1

-CH

Η

Η

5

1

Η

Η

6

6

Η

Η

5

5

Η

Η

very

Η

Η

very a c t i v e

C

3

H

- 2 5 -CH (CH ) 2

3

-a

2

<94

<55

3

Η

2

109

5

CH

3

Η

5

>120

CH

3

1

109

Η

+

0

Η

+

131

-C H

5

-C H

5

2

&

CH

-H 2

active

3

-CH -C H

2

OH OCH

3

CH

3

Only t e s t e d by p.o. route

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

198

TABLE V E f f e c t o f V a r y i n g the Atom that Bridges the Nucleus with the A c e t i c A c i d Moiety

CH C00H o

I.V. Dog X

2

3

R

-0-

H

Cl

CH

-0-

Cl

Cl

-S-

H

Cl

-s-

Cl

-so-

Na

+

Score

3

3

C H

5

6

CH

3

3

Cl

C H

5

6

Cl

Cl

C

-NH-

H

Cl

-NCH -

H

Cl

-CH -

H

Cl

3 CH

H

Cl

C H

3

2

-

2

2

H

2 5 CH 3

C H

2

+ + 0

3

0

5

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WOLTERSDORF E T A L .

(Aryloxy)aceUc

Acid

Diuretics

199

TABLE VI Effect

of Varying the Oxyacetic A c i d Group

R

I.V. Dog N a

+

Score

Τ

-

-CHFCOOH

6

-CH(CH )COOH

2

-C(CH ) COOH

+

<1

-(CH ) COOH

1

<17

-H

±

<26

+

6

3

3

2

2

3

-CH CONH 2

-CH COOC H 2

2

-CH S0 ~Na 2

2

a 5

+

3

-S0 N(CH ) 2

3

2

very

active

<4

6 a

2

active

12

-

a Tested

orally

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

200

DIURETIC

AGENTS

of them by methyl groups i s d i s a s t r o u s , as i s lengthening the chain by two methylene u n i t s . The phenol (R=H) and the acetamide (R=CH CONH ) have l i t t l e a c t i v i t y , but the e t h y l e s t e r i s f u l l y a c t i v e . Replacement of carboxy by s u l f o n a t e (R^I^SO^Na) gives a compound which i s h i g h l y a c t i v e i . v . but not p.o. Interestingly, the compound where R=S0 N(CH ) e x h i b i t s o r a l a c t i v i t y . The s t r u c t u r e / a c t i v i t y IS/A) data on the (acryloylphenoxy)a c e t i c a c i d s which we have presented i n d i c a t e that both s u b s t i ­ t u t i o n of the 6 - p o s i t i o n of the nucleus and i n t r o d u c t i o n of a methyl group on the carbon atom alpha to carboxy have d e t r i ­ mental e f f e c t s on a c t i v i t y . However, a number of patents (e.g., ^7, 8) have been assigned to Ciba-Geigy on benzofurans that can be viewed as 6 to α-cyclization products (Table VII) which v i o l a t e both of these S/A i n d i c a t i o n s . We prepared some examples of these s t r u c t u r e s , i n c l u d i n g th tw show i Tabl V I I d found the to be a c t i v e i n dogs, bu than the aromatic analog present) I t should be noted that these compounds have 6,7-dimethyl subs t i t u e n t s and that the analogous ( a c r y l o y l p h e n o x y ) a c i d i c a c i d s are l e s s a c t i v e than the corresponding d i c h l o r o compounds (see Table III)· Many s t u d i e s on the s i t e and mode of a c t i o n of e t h a c r y n i c a c i d have been reported. This compound belongs to the c l a s s of p o t e n t l o o p d i u r e t i c s , which i n c l u d e s furosemide and bumetanide, s i n c e i t s p r i n c i p l e a c t i o n i s i n the d i s t a l p o r t i o n of the loop of Henle (9-13). L i k e the m e r c u r i a l d i u r e t i c s (6), e t h a c r y n i c a c i d i s excreted p a r t i a l l y as the c y s t e i n e adduct (5) which has been shown to be a c t i v e i n man (14). The m e r c u r i a l s and e t h a c r y n i c a c i d a l s o show s i m i l a r e f f e c t s on decreasing the protein-bound s u l f h y d r y l groups i n r e n a l c e l l s i n dogs. No such e f f e c t was observed i n r a t s , a species i n which the drug i s v i r t u a l l y i n ~ a c t i v e (15-18). E t h a c r y n i c a c i d and i t s analogs are r e a d i l y c y c l i z e d to the corresponding indanones ( I ) , shown i n Table V I I I , which can be brominated (II) and s e l e c t i v e l y dehydrobrominated to the c o r r e ­ sponding indenones ( I I I ) or 2-alkylideneindanones (IV). Although T o p l i s s (19) had found a deschloro analog to be i n a c t i v e , com­ pounds b e a r i n g c h l o r i n e atoms i n the 6- and 7-positions were h i g h l y a c t i v e , as seen with the two compounds l i s t e d on the second l i n e of Table V I I I which are the p r e c i s e analogs of e t h a c r y n i c a c i d . This c o n s t i t u t e s the second exception to the i d e a that the 5- and 6-posiJions must remain u n s u b s t i t u t e d . Branching ( i . e . , where R and R are CH ) decreases a c t i v i t y i n both s e r i e s . A hypothesis regarding the behavior of the ( a c r y l o y l ­ phenoxy) a c e t i c a c i d s (V) i n body f l u i d s i s i l l u s t r a t e d i n F i g u r e 3 which shows t h e i r r e a c t i o n w i t h a v a r i e t y of s u l f h y d r y l c o n t a i n i n g compounds (VI), p a r t i c u l a r l y c y s t e i n e and g l u t a ­ thione, to form adducts (VII). Upon reaching the nephron, a r e a c t i o n occurs w i t h the receptor s u l f h y d r y l group (R SH, VIII) 2

2

2

n

lf

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WOLTERSDORF

ET AL.

(Aryloxy)acetic Acid Diuretics

TABLE V I I Benzofuran Analogs o f (Aeryloylphenoxy)acetic Acids

CH„

c

XT "

COÇ-R

2

3

Î COOH

COOH Double Bond Present -CH(CH ) 3

2

2

Dog Score

NO

2

•(CH ) CH

Iψ Na

3

YES

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

202

TABLE VIII C y c l i c Analogs of (Acryloylphenoxy)acetic ;i

Acids

0

(III)

XH C00H

CH C0OH 2

2

I.V.

1

Dog

Indenyloxyacetic Acid (III)

Na

+

(IV

>

Scores 2-Alkylideneindanyloxyacetic A c i d (IV)

R

R

H

H

4

-

4

4

3

2

-

1

6

-

CH

3

H

CH

0

CH

3

0

3 (CH )2

Ethacrynic Acid

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

11.

WOLTERSDORF E T

AL.

(Aryloxy)acetic Acid Diuretics

203

to g i v e a new adduct ( I X ) . For each compound, the d i u r e t i c a c t i v i t y w i l l depend, i n p a r t , on the e q u i l i b r i u m constants f o r r e a c t i o n 1 vs. r e a c t i o n 2 and the r e a c t i o n r a t e s . The most a c t i v e compounds w i l l be those whose e q u i l i b r i u m constants and r e a c t i o n r a t e s favor the receptor adducts ( I X ) . Confirmation of t h i s concept was attempted by preparing some mercaptan adducts of ethacrynic a c i d and studying t h e i r d i u r e t i c e f f e c t s i n dogs (Table I X ) . I t i s seen that the methyl mercaptan adduct has only weak a c t i v i t y , but that the c o r r e sponding s u l f o x i d e and sulfone are about as a c t i v e as e t h a c r y n i c a c i d . From these s e l e c t e d examples, i t w i l l be seen that a range of a c t i v i t i e s are obtained, from that as great as ethac r y n i c a c i d , i . e . , the c y s t e i n e and g l u t a t h i o n e adducts, to complete i n a c t i v i t y , i . e . , the mercaptoacetic and N - a c e t y l c y s t e i n e adducts. In the e t h a c r y n i a n a l y s i s were found to G. B. Smith and G. V. Downing proved to be q u i t e e n l i g h t e n i n g . They prepared the contour map shown i n F i g u r e 4 which shows the c o r r e l a t i o n of each compound's d i s t r i b u t i o n c o e f f i c i e n t (between chloroform and pH 7.4 b u f f e r ) with i t s r a t e constant f o r r e a c t i o n with mercaptoacetic a c i d . Each p o i n t shown on the map represents one compound with i t s i . v . dog d i u r e t i c score beneath the p o i n t . When e i t h e r d i s t r i b u t i o n c o e f f i c i e n t s or r a t e constants are considered s e p a r a t e l y , no c o r r e l a t i o n i s evident; however, when both parameters are considered and l i n e s drawn connecting p o i n t s of equal d i u r e t i c a c t i v i t y , order emerges. The map d e f i n e s regions or i s l a n d of each a c t i v i t y which provided guidance i n the design of compounds with maximal a c t i v i t y . ZF-(2-Haloacyl)phenox^7acetic Acids - Another c l a s s of potent d i u r e t i c s are those of the type shown i n Table X. The mosj a c t i v e members are those where R i s a l k y l or c y c l o a l k y l , R =H and X=bromo. Comparing those where R=isopropyl, the compound i n which X=Br i s s u p e r i o r to those where X=C1 or I . These compounds r e a c t with mercaptans, but some of the most d i u r e t i c members r e a c t very slowly and the products of the r e a c t i o n are d i f f e r e n t from those obtained with the acryloylphenoxyacetic a c i d s , i . e . , the X i s replaced by H, while the mercaptan i s o x i d i z e d to the d i s u l f i d e . ( V i n y l a r y l o x y ) a c e t i c Acids - About the time that the S/A r e l a t i o n s h i p s i n the ( a c r y l o y l p h e n o x y ) a c e t i c a c i d s e r i e s seemed c l e a r , compound 2 (Table XI) was prepared i n which the carbonyl and v i n y l groups were interchanged (20). The unexpected act i v i t y of t h i s compound suggested the s y n t h e s i s of the lower homolog (Compound 1) which was more a c t i v e , and the i n t r o d u c t i o n of a methyl group a t R which increased the a c t i v i t y (Compound 3 ) . The i n t r o d u c t i o n of a methyl at R on compound 2 markedly decreased the a c t i v i t y (compound 4 ) , but the c y c l i z a t i o n to the

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC AGENTS

204

TABLE IX Mercaptan Adducts of E t h a c r y n i c A c i d CI

CH C00H 2

I.V. Do R

Na

Score

R

Na

-SCH

3

2

-S0CH

3

6

-SCH CH(NHC0CH )C00H

+

-S0 CH

3

5

-SCH CH(NH )C00H

6

3

-SG (glutathione)

6-

?

-SCH 2

-SC(CH )

3

2

-S(CH ) C1

1

3

2

4

-SCH C00H

Score

0

2

2

3

2

2

-SC0CH -S)

3

2

4 2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WOLTERSDORF ET AL.

ι

R— C — C = C H „ C H 2

(Aryloxy)acetic Acid Diuretics

+

RSH

-_ ι . ^

0

Il

R—C—CHCH —SR 0

C H

5

2

(V)

(VI)

5

(VII)

+ W

2 R^H

(VIII)

^

^

R—Η^HCH —SR

1

2

(IX)

Figure 3. Reaction of (acryloylphenoxy)acetic acids with sulfhydryl compounds

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

206

TABLE X [4-(2-Haloacyl)phenoxy]acetic

Acids

k

CH COOH 2

R

R

-C H 2

5

1

l/

Η

Br

5

21

-CH(CH )

2

Η

Cl

+

No R

-CH(CH )

2

Η

Br

5

41

-CH(CH )

2

Η

I

2

Ό

Η

Br

6

Ο

Η

Br

3

X 3

3

C

H

- 2 5 -C H 2

5

C H

3

C H 2

5

>120 a

very a c t i v e

Br

+

21

Br

+

41

Active orally

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

11.

(Aryloxy)acetic Acid Diuretics

WOLTERSDORF E T A L .

207

TABLE XI (Vinylphenoxy)acetic Acids

Cl

I.V. Dog Compound

R

1 2

CH C03

H-

5

2 5 " CH C0-

H-

4

3

3

CH -

6

4

C H C0-

CH -

+

C

H

2

5

C O

3

5

3

-C0CH CH CH 2

6*

2

56 No Rx

No Rx Very Slow

6

CH C0-

CH C03

6*

2

7

CH C0 3

C H C0-

6*

2

8

C H C0-

C H C0-

3

2

5

5

4*

5

9

N0 -

H-

1*

<1

10

N0 -

CH -

6*

<1

11

N0 -

6*

2

12

N0 -

2 5" CH (CH ) -

*

8

13

CN-

H-

4*

No Rx

2

5

2

2

2

2

2

3

C

H

3

2

4

3

14

C H 00C-

C H 00C-

1

13

15

H NC0-

H NC0-

+

79

16

2

5

2

2

5

2

-C0CH(CH CH ) N2

2

2

3*

Very Slow

*Estimated from the response a t a dose below 5 mg/kg

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

208

AGENTS

corresponding cyclopentanone (Compound 5) g r e a t l y increased activity. Extending the s e r i e s (21) to those where both R and R are a c y l groups g r e a t l y enhanced the a c t i v i t y with maximal potency appearing when both groups are a c e t y l (Compound 6 ) . The compounds where R represents NO^ (22) a l s o were very a c t i v j . Increased potency was observed when the second group (R ) was methyl or e t h y l (Compounds 10 and 11). S i m i l a r but weaker a c t i v i t y was observed i n a number o f other compounds (23) i n which R and R represent a v a r i e t y o f electron-withdrawing groups, i . e . , cyano, ethoxycarbonyl, carbamoyl and sulfamoyl. The most a c t i v e compounds, i . e . , compounds 6-11, r e a c t the most a v i d l y with mercaptoacetic a c i d . In each s e r i e s , the other s t r u c t u r a l requirements f o r a c t i v i t y p a r a l l e l e d that found f o r the ( a c r y l o y l p h e n o x y ) a c e t i c a c i d s . One notable d i f f e r e n c mercaptan adducts whic a c e t i c a c i d s e r i e s . Thus, compound 6 and i t s mercaptoacetic a c i d adduct were equipotent, while the corresponding adduct o f e t h a c r y n i c a c i d was i n a c t i v e . O r a l s t u d i e s (Table XII) i n dogs on r e p r e s e n t a t i v e s from each s e r i e s i n d i c a t e that compound 3 i s four times as potent as furosemide, compound 6 i s 70 times and compound 10 i s 30 times as a c t i v e . We have discussed the three general types o f phenoxyacetic a c i d s shown i n Figure 5. They each can r e a c t with s u l f h y d r y l compounds, but each i n v o l v e s a d i f f e r e n t s i t e on the molecule as shown by the arrows. Our current concept i s t h a t , although these compounds can r e a c t with receptor s u l f h y d r y l groups and that such a r e a c t i o n c o n t r i b u t e s to t h e i r a c t i v i t y , t h i s r e a c t i o n i s not c r i t i c a l f o r s a l u r e s i s . A s e r i e s o f ( 2 , 3 - d i c h l o r o 4-saturated-acylphenoxy)acetic a c i d s , which cannot r e a c t with s u l f h y d r y l groups, were made and tested i n dogs (Table X I I I ) . Many o f these e x h i b i t e d a p p r e c i a b l e s a l u r e s i s . Particularly s i g n i f i c a n t are dihydroethacrynic a c i d (Compound 1) and the water adduct o f e t h a c r y n i c a c i d (Compound 4) which e x h i b i t e d weak, but s i g n i f i c a n t a c t i v i t y . Although these compounds exhibi t e d only modest a c t i v i t y , t h i s observation was o f profound t h e o r e t i c a l s i g n i f i c a n c e and s e t the stage f o r a major breakthrough i n the (acylphenoxy)acetic a c i d family o f d i u r e t i c s . To o b t a i n a complete p i c t u r e , the compounds that were made f o r subsequent s t u d i e s were evaluated o r a l l y i n chimpanzees, r a t s and dogs according to the c r i t e r i o n shown i n Tables XIV, XV and XVI. E v a l u a t i o n o f "dihydroethacrynic a c i d (Compound 1, Table XIII) i n chimpanzees revealed that i t had a weak but s i g n i f i c a n t s a l u r e t i c a c t i v i t y and t h a t , i n c o n t r a s t to e t h a c r y n i c a c i d , i t was u r i c o s u r i c . ( l - O x o i n d a n y l o x y ) a c e t i c Acids - Since 2-halo compounds, such as compound 1 i n Table XVII, had been used to synthesize the 1

w

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

WOLTERSDORF E T

AL.

(Aryloxy)acetic Acid Diuretics

209

TABLE XII O r a l Dog A c t i v i t y of (Vinylphenoxy)acetic Acids 6 h r . mEq.

Ave. Cmpd. No,

from

Table XI

mg/kg p.o.

Na

+

K

Dose

+

Excretion

οΓ

3

2

16

3

21

6

0.4

34

6

36

0.4

23

6

29

5

12

4

17

8

3

11

10

19

4

18

-

2

1

2

10 11 16 Furosemide

Placebo

0.4

O r a l t e s t s were c a r r i e d out on a colony of t r a i n e d female mongrel dogs weighing 8-10 kg. A l l dogs r e c e i v e d 100 ml of water the previous day and were f a s t e d overnight. On the day of the t e s t , 250 ml of water was administered o r a l l y , followed by 500 ml of water ( o r a l l y ) 1 hr l a t e r . One hour a f t e r the l a s t o r a l priming dose of water, bladders were emptied by c a t h e t e r i z a t i o n and the study was commenced by a d m i n i s t r a t i o n of compound or placebo. Compounds were given i n g e l a t i n capsules and the animals were maintained i n metabolism cages f o r c o l l e c t i o n of spontaneously voided u r i n e . Spontaneous u r i n e was combined with bladder u r i n e c o l l e c t e d by c a t h e t e r i z a t i o n at the end of 6 h r . Urine volumes were measured, and a l i q u o t s were analyzed f o r sodium, potassium and c h l o r i d e content by standard methodology. Values are reported as geometric means.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

Figure 5.

Site of reaction

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

WOLTERSDORF

ET AL.

(Aryloxy)acetic Acid Diuretics

TABLE X I I I (2,3-Dichloro-4-saturated-acylphenoxy)acetic

Acids

IH C00H o

Compd. No. 1 2

R

I.V.

-CH(CH C H 3

•

C H

C

2

Dog N a

1

H

3

-C(C H_)-OCH 1 2 5 2

3

4

-CH(CH OH)C H

3

5

-CH(CH CN)C H

ι

2

2

Score

2*

5

2 2 5 9

+

9

2

2

5

5

Ethacrynic Acid

2 6

*Estimated value from data a t 1 mg/kg

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

212

DIURETIC AGENTS

TABLE XIV Chimpanzee Data: E x c r e t i o n f o l l o w i n g 0.5 mg/kg i . v . o r 5 mg/kg p.o.

yEq/Min N a

+

Score

C C Δ Urate/ I n u l i n

0-49

0

0-.05

50-99

+

.05-.09

100-199

1

.10-.19

300-399

3

.30-.39

400-499

4

.40-.49

500 and Above

5

.50 and Above

200-299

Fasted, male chimpanzees weighing 21-77 kg were immobilized with p h e n c y c l i d i n e (which was shown not to a f f e c t the r e s u l t s ) (1.0-1.5 mg/kg i.m. plus 0.25 mg/kg i . v . as needed) and were prepared by c a t h e t e r i z a t i o n f o r standard r e n a l clearance s t u d i e s using r o u t i n e c l i n i c a l a s c e p t i c procedures. Pyrogenf r e e i n u l i n ( i . v . ) was used to measure glomeruluar f i l t r a t i o n r a t e (GFR). Clearance o f i n u l i n , urate and the e x c r e t i o n r a t e s of Na , Κ and CI were determined by standard Auto Analyzer techniques. ( I n u l i n and urate i n chimpanzee plasma are f r e e l y f i l t e r a b l e . ) Average c o n t r o l clearances were c a l c u l a t e d from three 20-min consecutive p e r i o d s . Drugresponse values were d e r i v e d as the average o f e i g h t 1520 min clearance periods a f t e r o r a l a d m i n i s t r a t i o n o f an aqueous s o l u t i o n o f the compound through an i n d w e l l i n g n a s a l c a t h e t e r . A l l data are reported as the d i f f e r e n c e between (average) treatment and c o n t r o l values obtained from s i n g l e experiments. +

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

WOLTERSDORF

ET AL.

(Aryloxy)acetic Acid Diuretics

213

TABLE XV 5 Hr. p.ο. Rat Scoring System

mEquivalents of N a at the Given mg/kg Dose +

Score

0

3

9

0

.3

.3

.3

.3

.3

+

.3

.3

.3

.5

.9

1

.3

.3

.4

.7

1.5

2

.3

3

Λ

.3

.9

1.9

2.9

4

.3

1.0

2.1

2.9

5

.3

1.7

2.9

3.4

6

.3

2.9

3.9

4.1

27

81

-

Female r a t s (Charles R i v e r , 150-170 g) were maintained over­ night on a sugar d i e t w i t h water ad l i b i t u m . The t e s t substance was d i s s o l v e d i n pure DMF and subsequently d i l u t e d with water (which contained 3 drops of Tween-80 per 100 ml) such that the f i n a l v e h i c l e was 4% DMF. At the time o f the t e s t , animals were given the v e h i c l e (as placebo) or t e s t substance suspended i n a f i n a l volume o f 5.0 ml p.o. Rats were housed i n groups of three i n metabolism cages. U r i n e was c o l l e c t e d f o r the 0-5 h r i n t e r v a l i n graduated c y l i n d e r s and was analyzed f o r sodium, potassium and c h l o r i d e content. Animals that r e c e i v e d placebo were run c o n c u r r e n t l y . R e s u l t s a r e r e p o r t e d as m i l l i e q u i v a l e n t s per cage and a r e the geometric means o f three cages per dose l e v e l . Standard methodology was used f o r determination o f e l e c t r o l y t e levels.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

214

AGENTS

TABLE XVI 5 Hr. p.o. Dog

mEquivalents

Scoring System

of Na

a t the Given mg/kg Dose

Score

0

1

2

5

10

20

0

2

2

2

2

2

2

+

2

2

2

2

8

>8

1

2

2

2

2

8

15

25

35

3

2

8

15

25

35

45

4

2

15

25

35

45

55

5

2

25

35

45

55

65

O r a l t e s t s were c a r r i e d out on a colony of t r a i n e d female mongrel dogs weighing 8-10 kg. A l l dogs r e c e i v e d 100 ml of water the previous day and were f a s t e d overnight. On the day of the t e s t , 250 ml of water was administered o r a l l y , followed by 500 ml of water ( o r a l l y ) 1 hr l a t e r . One hour a f t e r the l a s t o r a l priming dose of water, bladders were emptied by c a t h e t e r i z a t i o n and the study was commenced by a d m i n i s t r a t i o n of compound or placebo. Compounds were given i n g e l a t i n capsules and the animals were maintained i n metabolism cages f o r c o l l e c t i o n of spontaneously voided u r i n e . Spontaneous u r i n e was combined with bladder u r i n e c o l l e c t e d by c a t h e t e r i z a t i o n at the end of 5 h r . U r i n e volumes were measured, and a l i q u o t s were analyzed f o r sodium, potassium and c h l o r i d e content by standard methodology. Values are reported as geometric means.

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

WOLTERSDORF

(Aryloxy)acetic Acid Diuretics

ET AL.

215

TABLE XVII 2-Substituted-Indanones Cl

0

CH COOH 2

Compd No. 1

R

C

R

H

1

P.O. N a Rat

+

Score Dog

-Cl

3

1

-H

0

0

-H

P.O. Chimp Score Na Urate 2

+

2

- 2 5 -H

3

-CH

2

1

4

-Et

-H

2

1

4

+

5

-Pr

-H

+

1

5

1

6

-i-Pr

-H

2

3

5

2

7

-Bu

-H

+

1

1

1

3

8

-sec-Bu

-H

2

+

9

-t-Bu

-H

2

2

1

10

-Am

-H

1

1

1

+ + 2

11 12 13

--ο α -ο Ethacrynic Acid

-H

1

1

5

-H

1

1

2

3

-H

1

3

1

5

0 (retains urate)

0

6

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

216

DIURETIC

AGENTS

( i n d e n y l o x y ) a c e t i c a c i d s and the ( 2 - a l k y l i d e n e i n d a n y l o x y ) a c e t i c a c i d s described i n Table V I I I , the p o s s i b i l i t y that i t could f u n c t i o n as a pro-drug was considered. However, i t s s a l u r e t i c a c t i v i t y i n r a t s and i t s u r i c o s u r i c a c t i v i t y i n chimpanzees revealed i t t o be q u a l i t a t i v e l y d i s t i n c t from the m e r c u r i a l s and the unsaturated a r y l o x y a c e t i c a c i d s . I t was assumed that the observed b i o l o g i c a l d i f f e r e n c e s were due t o the unchanged compound, even though metabolic s t u d i e s i n d i c a t e d that some o f the expected dehydrohalogenation had occurred. T h i s l e d us t o prepare analogs o f compound 1 that were chemically more s t a b l e , such as compound 4, which i s an annulated form o f "dihydroethac r y n i c a c i d " (24-27). I t s a c t i v i t y p r o f i l e was very s i m i l a r t o that o f compound 1. The r e p r e s e n t a t i v e compounds o f Table XVII e x h i b i t a range of s a l u r e t i c and u r i c o s u r i whe R represent variety of alkyl, cycloalky i s o p r o p y l analog (Compoun ) appeare optimum a c t i v i t y . The e f f e c t o f s t r u c t u r a l m o d i f i c a t i o n o f the nuclear s u b s t i t u e n t s and r e o r i e n t a t i o n o r v a r i a t i o n o f the oxyacetic a c i d moiety was about the same as had been observed i n the ( a r y l o x y ) a c e t i c a c i d s e r i e s . A most s i g n i f i c a n t c o n t r i b u t i o n t o the a c t i v i t y o f these ( i n d a n y l o x y ) a c e t i c a c i d s (27-28) occurred with the i n t r o d u c t i o n of a second 2-substituent (Table XVIII). These compounds were g e n e r a l l y considerably more a c t i v e than the corresponding monosubstituted compounds. To q u a n t i t a t e these comparisons i n r a t s , dose-response curves with high s t a t i s t i c a l s i g n i f i c a n c e were developed. The numbers i n parenthesis i n Table XVIII are the f o l d g r e a t e r a c t i v i t y found f o r each 2-R, 2-CH- compound as compared t o the monosubstituted analog (Compound 17. Optimal d i u r e t i c a c t i v i t y was seen with compounds 4 and 6 (MK-196), but the l a t t e r compound e x h i b i t e d optimal dual ( d i u r e t i c - u r i c o s u r i c ) a c t i v i t y . Increasing the s i z e o f R t o e t h y l (Compounds 7 and 8) was detrimental to d i u r e t i c a c t i v i t y . Since u r i c o s u r i c and d i u r e t i c a c t i v i t i e s do not run p a r a l l e l , some compounds which are very weak d i u r e t i c s , e.g., (2-ethyl-3-phenyl-6,7-dichloro5-indanyloxy)acetic a c i d , e x h i b i t potent u r i c o s u r i c a c t i v i t y . S u b s t i t u t i o n on the phenyl group o f MK-196 had a marked e f f e c t on d i u r e t i c and u r i c o s u r i c a c t i v i t y (Table XIX). Although there was considerable species v a r i a t i o n , the p - f l u o r o d e r i v a t i v e (Compound 4) showed the best o v e r a l l a c t i v i t y . Replacement of the phenyl group of MK-196 by a 2 - t h i e n y l group gave a compound which was equipotent to MK-rl96. Replacement o f the carboxy group o f MK-196 by a number of groups, i n c l u d i n g 5t e t r a z o l y l , u s u a l l y produced compounds with considerably decreased s a l u r e t i c a c t i v i t y . Numerous s t r u c t u r a l v a r i a t i o n s were made i n p o s i t i o n s 1, 2 and 3 o f the indane r i n g (29.). Reduction o f the 1-oxo group o f MK-196 t o hydroxy provided two diasteriomers, both o f which were

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

WOLTERSDORF

11.

(Aryloxy)acetic Acid Diuretics

ET AL.

217

TABLE XVIII (2,2-Disubstituted-Indanyloxy)acetic Acids

ο

Cl

X

CH COOH 2

Compd. No.

R

R

1

Rat

(FOLD χ Compd. 1)

P.0. Chimp Scores Na Urate +

Dog

1

-CH

3

H-

2

(1)

1

2

-CH

3

CH -

3

(4)

1

3

+

3

-Et

CH -

3

(15)

3

5

1

4

i-Pr

CH -

3

(53)

3

4

+

5


CH -

2

(5)

2

2

1

(50)

3

5

3

2

1

6 7 8 9

3

3

3

-o -a

3

CH 3

C

-o

H

2

H

1

+

3

1

2

1

3

1

2 5-

C

2 5-CH CH CH CH 2

2

2

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

218

TABLE XIX E f f e c t of Substituents on the Phenyl Group o f MK-196 Cl

Compel. No.

Q

R

1

H

3

3

5

3

2

4-Br

1

+

+

3

4-C1

+ +

1

3

2

4

4-F

2

3

5

4-0CH

2

6

4-OH

+

2

7

3-0H

1

2

3

3

4

4

+

1

+

8

4-NH

2

1

1

2

+

9

4-N0

2

4

2

1

+

5

5

+

5

+

10

4-^S0 NH

2

1

11

4-COCH

3

+

2

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

11.

WOLTERSDORF E T A L .

(Aryloxy)acetic Acid Diuretics

219

about as a c t i v e as the parent (30). A l k y l or a r y l groups i n the 3 - p o s i t i o n u s u a l l y decreased s a l u r e t i c a c t i v i t y , and e i t h e r d i d not a f f e c t or increased the u r i c o s u r i c a c t i v i t y . A 3-oxo subs t i t u e n t g e n e r a l l y provided compounds with a c t i v i t i e s only s l i g h t l y below that of t h e i r parents (31). The i n f l u e n c e o f the c h i r a l center a t the 2 - p o s i t i o n can be seen from the study i n v o l v i n g the four enantiomeric p a i r s (Table XX). Although there was some v a r i a t i o n among s p e c i e s , the enantiomeric e f f e c t was most pronounced with compounds 1 and 4. Four of the ( i n d a n y l o x y ) a c e t i c a c i d s have been s t u d i e d clinically: compounds 6 and 11 from Table XVII and compounds 5 (MK-473) and 8 (MK-196) from Table XVIII. The r e l a t i v e s a l u r e t i c and u r i c o s u r i c potencies i n man were p r e d i c t a b l e from the animal data, i . e . , MK-196>MK-473>compound ll>compound 6 (Table XVII). MK-196 has r e c e i v e d extensiv pharmacological biochemical and c l i n i c a l e v a l u a t i o hypertensive e f f e c t i n mg/kg. I t was more potent than furosemide or h y d r o c h l o r o t h i a z i d e when given d a i l y f o r three days. I t a l s o was a n t i h y p e r t e n s i v e i n r e n a l hypertensive monkeys but not i n r e n a l hypertensive dogs. MK-196 i s w e l l absorbed i n mice, r a t s , dogs, monkeys, chimpanzees and humans (34-36). In r a t s and dogs, there was l i t t l e metabolism o f the drug, which i s excreted mainly i n the feces. In mice and monkeys, there was some metabolism and about equal e l i m i n a t i o n by the f e c a l and u r i n a r y routes. In chimpanzees, the major e l i m i n a t i o n i s v i a the u r i n e and, i n man there i s considerable metabolism, p r i m a r i l y to the 2-(p-hydroxyphenyl) d e r i v a t i v e (Compound 6, Table XIX). In chimpanzees (37-39), the u r i n a r y e x c r e t i o n o f MK-196 i s increased t e n - f o l d when the u r i n e i s a l k a l i n e . The drug i s s e c r e ted by the r e n a l tubule and p a s s i v e l y b a c k - d i f f u s e s across the tubular e p i t h e l i u m by a pH-dependent process. Since i t i s known that urate i s reabsorbed and secreted i n the proximal tubule, the s t u d i e s i n chimpanzees (38) suggested that the a c t i o n of MK-196 on the clearance of urate i s l o c a l i z e d at t h i s s i t e . Micropuncture s t u d i e s i n r a t s (40) i n d i c a t e that the drug a f f e c t s e l e c t r o l y t e r e a b s o r p t i o n i n the loop of Henle and c o l l e c t i n g duct, while concentration gradient s t u d i e s i n dogs (42) i m p l i c a t e s i t e s i n the proximal tubule and ascending loop o f Henle. Biochemical s t u d i e s by Kuehl (43) and h i s colleagues i n d i c a t e that MK-196 and other compounds i n t h i s s e r i e s owe a t l e a s t p a r t of t h e i r a c t i v i t y to t h e i r e f f e c t s on p r o s t a g l a n d i n metabolism. MK-196 and i t s enantiomers (Table XXI) i n h i b i t PGE-9-ketoreductase, the enzyme that converts PGE to PGF . To a l e s s e r extent, i t i n h i b i t s PG-15-hydroxydehydrogenase, tSe enzyme which d e a c t i v a t e s p r o s t a g l a n d i n s . MK-196 has a greater i n h i b i t o r y e f f e c t on these enzymes than the c l a s s i c a l d i u r e t i c s as shown i n Table XXI. The o v e r a l l e f f e c t would be to i n c r e a s e the r e n a l l e v e l s of PGE . 2

n

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

220

AGENTS

TABLE XX Enantiomers o f (Indanyloxy)acetic Acids

Compd. No. 1 2

1

R

R

-Q -a

1

H

3

-CH(CH )

4

-Q

3

2

CH

3

CH

3

Furosemide Hydrochlorothiazide

Enantiomer

Rat

Dog

Na

+ + + + -

2 1

2 1

3 2

2 1

3 2

2 1

2 2

1 2

2 3

3 5

5 4

4 1

1 3

1 2

1 6

1 2

3

5

5

0*

2

2

1

0*

Probenecid *Decreased E x c r e t i o n tDose = 10 mg/kg

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Urate

It

11.

WOLTERSDORF

ET AL.

(Aryloxy)acetic Acid Diuretics

221

TABLE XXI E f f e c t o f MK-196 on P r o s t a g l a n d i n Metabolism

I D

50

μΜ

Compd.

Enantiomer

9-Ketoreductase

MK-196

+

15

178

MK-196

+

50

351

Ethacrynic Acid

307

709

Furosemide

499

2419

>2514

>2514

a

15-Hydroxydehydrogenase

MK-196 Mersalyl Acid

Hydrochlorothiazide

^Measured by determining the enzymatic conversion of H-PGE^ to H-15-keto-PGE i n the presence of NADP. Measured by the method of Oien e t a l \43).

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

222

DIURETIC AGENTS

In man, MK-196 (44-46) was found to be a w e l l t o l e r a t e d s a l u r e t i c - d i u r e t i c . An o r a l dose of 10 mg e l i c i t e d a t o t a l 24 hour s a l u r e s i s equivalent to that of 40 mg of furosemide. A d m i n i s t r a t i o n of furosemide caused a s i g n i f i c a n t i n c r e a s e i n plasma urate l e v e l s , whereas the continued use of MK-196 caused l e s s i n c r e a s e i n plasma urate l e v e l s . D a i l y o r a l a d m i n i s t r a t i o n of e i t h e r 10 or 15 mg of MK-196 lowered the blood pressure of hypertensive p a t i e n t s as much as 50 mg of h y d r o c h l o r o t h i a z i d e did. This drug i s c u r r e n t l y undergoing extensive c l i n i c a l trials. Indeno/3 >4-b7furan-2-carboxylie Acids - The i n t e r e s t i n g a c t i v i t y e x h i b i t e d by the benzofuran analogs of the (acryloylphenoxy)a c e t i c acids o f the type seen i n Table V I I suggested the synthesis of some indeno/3 of the a c t i v e ( i n d a n y l o x y ) a c e t i these compounds are potent d i u r e t i c s (47, 48). In t h i s s e r i e s , u r i c o s u r i c a c t i v i t y was e x h i b i t e d only by those compounds where R was phenyl. Since these compounds have two c h i r a l centers, two diasteriomers are p o s s i b l e , and i n one instance both d i a — steriomers (Compounds 3 and 4) were obtained and shown to have q u i t e s i m i l a r d i u r e t i c and u r i c o s u r i c a c t i v i t i e s . (Acylphenoxy)acetic Acids (Table XXIII) - The observation of s a l u r e t i c and u r i c o s u r i c a c t i v i t y i n t i e n i l i c a c i d (Compound 1, Table XXIII) and some of i t s analogs (49) and i n "dihydroe t h a c r y n i c a c i d " (Compound 2, Table XXIII) (26) revealed that pharmacodynamic a c t i v i t y e x i s t e d i n s e v e r a l (2,3-dichloro-4acylphenoxy)acetic a c i d s . The f a c t that a c t i v i t y a l s o was observed i n compound 3 and a number of r e l a t e d s t r u c t u r e s i n d i c a t e d that the p o r t i o n of the molecule represented by R could be a l k y l , h e t e r o c y c l i c , a r y l or a r a l k y l . Since annulation of ( a c r y l o y l p h e n o x y ) a c e t i c a c i d s and ( i n d a n y l o x y ) a c e t i c acids t o the corresponding dihydrobenzofurans or dihydroindeno^5,4-b/furans e i t h e r maintained or improved a c t i v i t y , an analogous a n n u l a t i o n of r e p r e s e n t a t i v e ( a c y l phenoxy) a c e t i c a c i d s was c a r r i e d out (50) as recorded i n Table XXIV. Studies i n v o l v i n g compound 1 i n r a t s , dogs and chimpanzees revealed i t to be 40 to 100 times as potent a d i u r e t i c as t i e n i l i c a c i d with about equal a c t i v i t y as a u r i c o s u r i c agent. Most i n t e r e s t i n g i s the f a c t that compound 1 i s a racemate which upon r e s o l u t i o n a f f o r d s two enantiomers i n which there i s a complete s e p a r a t i o n o f pharmacodynamic p r o p e r t i e s . The minusform possesses only u r i c o s u r i c a c t i v i t y , while the p l u s enantiomer possesses only d i u r e t i c p r o p e r t i e s . This permits the formulation of combinations o f the enantiomers to obtain any d e s i r e d r a t i o of d i u r e t i c to u r i c o s u r i c a c t i v i t y . I t i s noteworthy that compounds i n which R represents a v a r i e t y of a r y l , a r a l k y l , or h e t e r o c y c l i c groups r e t a i n the a c t i v i t y e x h i b i t e d by

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

(Aryloxy)acetic Acid Diuretics

WOLTERSDORF E T A L .

11.

223

TABLE XXII Indeno[5,4-b]Furan-2-Carboxylic Acids

Compd. No.

1 R

1

R

-(CH

2

CH

3

3

(a)

CH

3

4

(3)

CH

3

2

\~

<]

-o

P.O. N a Rat

+

Scores Dog

P.O Na

Chimp Scores Urate

5

4

5

0

4

4

3

0

3

5

5

5

4

6

6

4

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

224

AGENTS

TABLE XXIII (4-Acylphenoxy)acetic Acids

\H COOH O

Compd. No.

P.O. Rat Scores

R

1

-ώ

2

-CH(CH )C H

3

3

2

5

I.V. Dog Scores

Na Δ mEq/min

çUrate Inulin

1

1

434

.376

+

1

400

.213

+

1

363

.309

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

11.

WOLTERSDORF E T A L .

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Acid

Diuretics

225

TABLE XXIV 4-Acyldihydrobenzofurans

r

1

?

COOH

P.O. B i o l o g i c a l A c t i v i t y Scores Compd. No.

1

Rat

Enantiomer

+ + -

-û -ô -ώ -ô T?

Diuretic Dog Chimp

Uricosuric Chimp

5

4

5

3

0

0

0

5

6

5

5

0

3

3*

3

3*

5

1

2

+

3

+

4

+

3

4

2

5

+

3

5

3

6

+

7

T i e n i l i c Acid

3

4

*I.V.

-^K)CH

3

3 1

+

Scores

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

DIURETIC

226

TABLE XXV Reduction Products of 5-Acylbenzofuran-2-Carboxylic CI

Acids

X

COOH

P.O. B i o l o g i c a l A c t i v i t y Compd. No.

Diuretic Dog Chimp

Scores

Uricosuric Chimp

X

Rat

1

0

4

4

4

3

2

H (OH)

1

4*

5*

1

+

4

2*

1

3

H

2

*I.V. Data

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

AGENTS

11.

WOLTERSDORF E T A L .

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Diuretics

227

TABLE XXVI COLLABORATORS IN THE PHENOXYACETIC ACID RESEARCH

ORGANIC CHEMISTS A. AUGENBLICK J . J . BALDWIN J.B. BICKING W.A. BOLHOFER A.A. DEANA

N.P. C.N. W.J. W.F.

GOULD HABECKER HOLTZ HOFFMAN

J.K. S.F. J.W. F.C.

CM. E.M. J.M. G.É. T.P

HORNER KWONG MASON NOVELLO

ROBB SCHULTZ SPRAGUE STOKKER STROBAUGH

PHYSICAL AND ANALYTICAL CHEMISTS B.H. ARISON E.L. CRESSON

G.V. DOWNING Y.C. LEE

W.R. MCGAUGHRAN W.C. RANDALL

G.B. SMITH K.B. STREETER

BIOCHEMISTS D.E.

DUGGAN

E.H.

HAM

F.A. KUEHL

H.G. OIEN

PHYSICIANS G.H. BESSELAAR R.O. DAVIES

Z.E. DZIEWANOWSKA

J.J.

SCHROGIE

K.F. TEMPERO

BIOLOGISTS J.E. BAER K.H. BEYER D.L. BOHN

C H . DUNCAN R.M. EVANS C M . FANELLI

E.K. MAZACK J.E. MICHAELSON H.F. RUSSO

L.S. WATSON (Dec.) T.I. WISHOUSKY A.C ZACCHEI

ACKNOWLEDGMENTS A.

SCRIABINE

R.F. HIRSCHMANN

C.A. STONE

In Diuretic Agents; Cragoe, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

228

DIURETIC AGENTS

compound 1. Of the many m o d i f i c a t i o n s o f the s t r u c t u r e o f the benzofurans that have been made, one o f the more i n t e r e s t i n g i n v o l v e s that o f the carbonyl group. Reduction to the hydroxymethylene (one diasteriomer) o r to methylene produced compounds which maintained good a c t i v i t y i n dogs and chimpanzees but not i n r a t s (see Table XXV). In summary, the r a t i o n a l design o f d i u r e t i c s based upon e i t h e r the s t r u c t u r a l f e a t u r e s or mechanism o f a c t i o n o f the m e r c u r i a l d i u r e t i c s l e d to the discovery o f about a dozen new types o f potent d i u r e t i c s . One o f these, e t h a c r y n i c a c i d , has become a c l i n i c a l l y u s e f u l drug and s e v e r a l others show s i m i l a r promise. We wish to pay t r i b u t e to the chemists, b i o l o g i s t s and p h y s i c i a n s l i s t e d i n Table XXVI who c o n t r i b u t e d t o these s t u d i e s which were conducted ove

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