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UniversiV MIcixSilms International 300 N. Z eeb Road Ann Arbor, Ml 48106

8311751

Hassan, Ahmed Mostafa-Mi

INDANPROPIONIC ACID PGF(2ALPHA) ANTAGONISTS

The Ohio State University Ph.D. 1983

University Microfilms I ntern sti G nal 300 n . zeeb Road, Ann Arbor, MI 48106

INDANPROPIONIC ACID PCF 2a ANTAGONISTS

DISSERTATION

Presented In Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

Ahmed M M . Hassan, B.S., M.S.

*****

The Ohio State University

1983

Reading Committee:

Donald T. Witiak, Ph. D. Duane D. Miller, Ph. D. Ralf G . Rahwan, Ph. D . Larry V. Robertson, Ph. D. ✓~>Appr oved

Adviser College of Pharmacy To Those Who Made it Possible,

My Parents, My Wife and My Educators

ii ACKNOWLEDGMENTS

I wish to express my sinscere appreciation to my adviser. Professor Donald T. Witiak, for his valuable guidance, encouragement, understanding, and financial support throughout this work.

I wish to thank Professor Ralf G. Rahwan, for helpful discussions and for facilitating the biological evaluation of the compounds and for financial support on National Institute of Health grant No. HD-14853.

I wish to thank Dr. Richard J. Heaslip and Dr. Franziska R. Del Vecchio for carrying out the biological evaluation on these compounds. Special thanks are due to Mr. Jack Fowble and Miss. Anna Liao who provided assistance in NMR and mass spectroscopy technology and to Mr. Nicholas E. Felt who provided considerable assistance to me in the library.

Finally I am particularly grateful to my parents and my wife Sarnia for their love and encouragement during the course of these studies.

iii VITA

January 17, 1950 ...... Born- Alexandria, Egypt.

1968-1973 ...... B.S.(hons.), University of Alexandria.

1977 ...... M.S. University of Alexandria.

1978-1980 ...... Teaching Associate, Department of Medicinal Chemistry, The Ohio State University, Columbus, Ohio.

1980-1983 ...... Research Associate, Department of Medicinal Chemistry, The Ohio State University, Columbus, Ohio.

PUBLICATIONS

Ragab M. Shafic, Raafat Soliman and Ahmed M. M. Hassan New Biphenylyl Derivatives:l-(A-Biphenylyl)-2-phenylethylamines : Potential Antispasmodic and Cardiovascular Agents, J. Pharm. Sci. 991, 1978.

Richard J. Heaslip, Ralf G. Rahwan, Ahmed M. M. Hassan and Donald T. Witiak Uterine Relaxant effects of Mono- and Di- benzyloxyindanpropionic Acids Res. Comm. Chem. Path, and Pharm. 32, 251, 1981.

FIELDS OF STUDY

Major Fields: Pharmaceutical Organic Chemistry Medicinal Organic Chemistry

Iv TABLE OF CONTENTS

page

DEDICATION ...... il

ACKNOWLEDGMENTS ...... ill

VITA ...... iv

LIST OF FIGURES ...... vi

LIST OF TABLES ...... vii

INTRODUCTION ...... 1

HISTORICAL BACKGROUND ...... 4

PROSTAGLANDIN ANTAGONISTS ...... 21

STATEMENT OF THE PROBLEM ...... 44

RESULTS AND DISCUSSION ...... 52

EXPERIMENTAL SECTION ...... 58

PHARMACOLOGY ...... 74

REFERENCES ...... 78 LIST OF FIGURES

Figure Page

1. Bioconversion of Linoleic Acidto Prostaglandin .. 10

2. Prostaglandin Biosynthesis ...... 11

3. A Probable Relationship between Prostaglandin and

Physiological Effect ...... 17

4. Schematic Representation of the Synthesis of

Prostaglandin in the LH-responsive Cell...... 20

5. The Hairpin and L-shaped Conformation of PGF2 q

and PGB^ ...... 47

vi LIST OF TABLES

Table Page

1. Effect of Compounds 44 . 45 and 5_1_ on PGF^g-lnduced

Contractions of The Mouse Uterus ...... 76

vii Chapter I

INTRODUCTION

Premature labor and spontaneous abortion are the most common pathological complications of pregnancy. It is estimated that about 1 0 % of the total number of pregnancies end in spontaneous abortions despite available therapy.

Existing uterine relaxants lack selectivity for uterine smooth muscle. Thus, adrenergic g^-receptor agonists, while effectively relaxing the pregnant uterus, may exert adrenergic effects elsewhere. Furthermore, the uterine relaxant effect of alcohol is not without its technical difficulties, and the value and safety of progestational drugs for the maintenance of complicated pregnancies have recently been questioned. Consequently, there is considerable interest in the development of new and more selective uterine relaxants.

Effects of prostaglandins on the female reproductive system are particularly striking. PGF2 Q appears to 1-3 participate in the etiology of spontaneous abortion » aod inhibitors of prostaglandin synthetase (cyclooxygenase) have recently been reported to be effective and relatively safe 3 in the prevention of spontaneous abortion in women • There is a need for development of a specific primary

- 1 - 2

prostaglandin antagonist. The variety of responses of

pulmonary and systemic arterial pressure of PCs suggests

that these responses are mediated by different PG

receptors*. Salicylates and other nonsteroidal, anti

inflammatory compounds block the synthesis of all endogenous

PCs by inhibiting the enzymatic oxygenation and cyclization

of arachidonic acid^* These agents do not differentiate the

responses of different prostanoic substances.

Antagonists specific for each PG species would aid

identifying a receptor mechanism. However, few potent,

selective PG antagonists have been identified in vivo to

date^. A pharmacological antagonist should be specific,

potent, reversible and possess no agonist activity. The

objective of this work was to develop specific primary PG

antagonists which:

(1 ) could serve as molecular probes in the elucidation of

the roles of PG agonists in physiological and pharmacologi

cal processes and;

(2 ) may be useful therapeutic agents in the treatment of

various disorders.

The original series of 2-indanpropionic acids^ which

provided the impetus for the present study involved

2 -propyl-5 ,6 -dibenzyloxy-l-oxo-2 -indanpropionic acid (J^) which was found to selectively and reversibly inhibit PGp2:j — 5 on the isolated mouse ileum at a concentration of 3x10 M. This compound exhibited significantly less inhibitory activity against acetylcholine- (AcCh) or potassium chloride-induced contractions. Higher concentrations of ^ -4 (10 H or greater) significantly inhibited the contractions induced by PGF2 Q , AcCh and potassium chloride.

C 0 2 H o Chapter II

HISTORICAL BACKGROUND

In 1930 Kurzrok and leib^ found that human semen both relaxed and contracted human uterine muscle strips. From these observations, they inferred the presence in semen of

8 9 two substances, one contractile, the other relaxant * • A few years later Goldblatt^^ isolated a depressor like substance from human seminal plasma that contracted isolated rabbit intestine and isolated guinea-pig uterus. At about

T 1 12 the same time, von Euler * found that similar activity was displayed by extracts of prostate glands and seminal 13 lA vesicles of several animal species. In 1935 von Euler * coined the name "prosaglandin" for the substance, known to be an acidic lipid. An extensive description of the pharmacological properties of this substance was later published.

Over the next 30 years, interest in prostaglandins declined. However, the isolation, characterization and synthesis of representative compounds in the early 1960s generated a renewed interest. Prostaglandins have been detected in virtually every tissue and body fluid; their production increases in response to numerous diverse stimuli. These substanses produce, in minute amounts, a

- 4 - 5 broad spectrum of effects that embrace practically every biological function. Inhibition of PG biosynthesis is now recognized as the mechanism by which nonsteroidal anti inflammatory drugs such as aspirin exert their effects. In

1964 Bergstrom and coworkers^^ ’ and van Dorp and associ- 17 18 ates * independently achieved the biosynthesis of PGEg from arachidonic acid using homogenates of sheep seminal vesicles.

Nomenclature

Prostaglandins are a family of closely related fatty acids containing a cyclopentane ring with two adjacent side chains, one of which has a carboxyl group at a terminal position. The naturally occuring prostaglandins may be regarded as derivatives of prostanoic acid 2 » whose carbon atoms are numbered as shown. Although they may be named by their relationship to prostanoic acid, the natural prostaglandins are usually divided into five groups and are referred to by the letters A, B, C, E and F.

All five groups feature a hydroxyl function at and a trans double bond at the 13, 14 position. The E and F series are frequently referred to as primary prostaglandins.

Both possess an additional hydroxyl group at The E series contains a carbonyl group at Cg, whereas the F series has a hydroxyl group at that position. The A, B and C series may be regarded as cyclopentene dehydration products of the E series. These three series lack a hydroxyl group at Cii, but possess a double bond in the ring.

ÔH

PGEj (3)

C O ^ H C O 2 H

OH OH

PGEj U)

CO 2 H CO 2 H

PGA, (2)

■ C O 2 H

PGEj (B) PGCj (9 ) 7

Each of these five series is subdivided on the basis of

the number of additional side chain double bonds present.

The total number of double bonds is indicated by a subscript

numeral after the letter denoting the series. Therefore,

prostaglandin (PGE^j contains only the double

bond. PGE2 has an additional cis double bond at C5 g and

PGEg possesses a third double bond at ^17 18* These

additional side chain double bonds occur in the same

positions and with the same configuration for all five

series, although prostaglandins PGA3 , PGBg, and PGCg have

never been isolated from natural sources.

Since all naturally occuring PGs isolated to date have

the (X configuration at Cjj and this designation is

normally omitted in abbreviated forms. In the F series a erg

are used to denote the configuration at Cg.

To denote various stereochemical changes differing

terms are employed. For example, if the two side chains bear a cis relationship to one another, the compounds are

termed 8 -isoprostaglandins (e.g., 6 -iso-PGF - [ r v )• Hydroxyl groups in the unnatural p-configuration at or are referred to as epi (e.g., 15-epi-PGEp). In addition, the

hydroxyl group may be named after the Cahn-Ingold-Prelog convention, i.e. 2 for theq and ^ for the p form.

Natural PGs are levorotatory. Their dextrotatory enan- tiomers are known by the prefex ent (e.g., ent-PGE^). lUPAC nomenclature for PGE^ 1s (11a , 13^,15^)-11,15 —dihydroxy-

9-oxoprost-l3-en-l-oic acid. OH ÔH OH

8-lso-PGE^ (10) 11-epi-PGE^ (JJ^)

O ^ H

OH OH ÔH

■nt-PGEi (12) PGFip (13)

Examples of Stereochemlchal Variations

of the Natural Prostaglandins 9

Prostaglandin Biochemistry

19 PGs are present In many mammalian tissues . Even

though relatively large amounts of PGs can be detected In

only a few tissues, the capacity for PG biosynthesis can be

demonstrated in all tissues except red blood cells. PGs are

rarely stored in tissues. Usually PGs are synthesized

rapidly, from tissue stores containing fatty acid precursors 20 in response to local stimulation • PG synthesis can be

initiated by mechanical (stretch or distension), chemical

(catecholamines, serotonin, histamine, AcCh, glucagon)j or 21 neuronal stimulation.

In 1930 it was discovered that linoleic acid (1_4) is a 2 2 necessary element in the diet of healthy rats • Lack of 14

caused poor growth, skin lesions, kidney damage, and 22 23 sterility * . This finding was later extended to other

species. Including humans. Subsequently, It was recognized

that many tissues convert linoleic acid (j^) to gamma-

linolenic acid (15) ; this intermediate afforded dihomo-

gamma-linolenic acid (1_6^) and arachidonic acid (17).

Presumably, these fatty acids are essential because they are

obligatory precursors of PGs^^’^^*^^ (Figure 1).

In man, arachidonic acid (1^) is either derived from

dietary linoleic acid (1_4) or is Ingested as a constituent

of meat. Estérification and incorporation of a phospholipid component of a cell membrane or other complex blo-lipld represents the physical state of PG in cells. Arachidonic 10

acid is released from membrane phospholipids by the action

of the enzyme phospholipase A g . Once released, arachidonic

acid is rapidly metabolized to oxygenated products by

distinct enzymatic mechanisms, a cyclooxygenase and a llpo-

oxygenase (Figure 2).

Linoleic acid (14) 1

gamma-Linolenic acid (15)

CO 2 H

Dihomo-gamma-Linolenic acid (lj6 ) _____ ^ PG^ series

Arachidonic acid (17) PG2 series

Figure 1. Bioconversion of Linoleic Acid to Prostaglandins 11

Arachidonic acid (17)

Fatty acid

cyclooxygenase

COOH

Lipooxygenase

COOH

bOH 0__ V . 0-0

12-HPETE (20)

I2-Hydroperoxy-5,8,10,14" 0 COOH eicosatetrenoic acid

I 0 bOH COOH

PGGj (18)

'■COOH 12-Hydroxy-5,8,10,14- eicosatetrenoic acid (2 1 )

6h PGH2 (19)

PGEg (4) PGF2q<5)

Figure 2. Prostaglandin Biosynthesis 12

P rostaglandln Pharmacology

PGs display a wide diversity of biological

effec. They stimulate contraction and relaxation of

smooth muscle, secretion (including some endocrine gland

secretions), and blood flow. These individual effects, how

ever, may be quite specific for a given PG. For example j

PGF2q; raises blood pressure, while PGE2 lowers blood

pressure.

PG concentrations may be critical to peptic ulcer

development. Thus, PCE^ or PGE2 can inhibit gastric

secretions in dogs. Furthermore, these same PGs prevent

gastric and duodenal ulcers in rats.

PGs exert powerful actions on platelets. Some of them,

like PGE^^ are inhibitors of human platelet aggregations in

vitro at concentrations of 0 . 1 pM. PGE2 exerts variable

effects on platelets and is a potentiator of aggregation at

low concentraions (below 1 pM) and an inhibitor at higher

concentrations.

Both PGEj and E2 are powerful vasodilators. When given

intraarterially, they cause a fall in blood pressure which is accompanied by a reflex increase in heart rate owing to a direct dilation of vascular smooth muscles. The effect is not inhibited by a or p adrenergic agents, antihistamines, 36 37 LSD, ganglionic blocking agents, or vagotomy ’

PGs contract or relax many smooth muscles besides those of the vasculature. Response may vary with species, type of 13

P G , endocrine status of the tissue, and experimental

condition. Few smooth muscles are unaffected by PGs and may

display intense and consistent responses. In general, PGF

contracts and PGE relaxes bronchial and tracheal muscle from

various species, including man. Asthmatic individuals are

particularly sensitive wherein PGF2Q; causes intense bronchospa sm. In contrast, both PGE^ and E 2 are potent bronchdilators when given to such patients by aerosol; the potency of PGE^ may exceed that of isoproterenol^® ^ ^ «

PGs E and F produce strong contraction of isolated guinea pig uteri in estrus or diestrus. The contractile response is most prominent before menstruation. Uterine strips from pregnant women are uniformally contracted by PGE and PGF. In contrast to uterine behavior in vitro, the human uterus in vivo, whether pregnant or not, is always contracted by intravenously administered PGE^, PGE2 and PGF2Q.

The response is prompt and dose dependent.

Many stimulant and depressant effects of PGs on the 41—43 central nervous system (CNS) have been reported • PGEs cause sedation, stupor, catatonia and behavioral changes following injection into the cerebral ventricles of cats.

To elicit such effects higher concentrations of PGs are required.

In man, PGEs cause pain when injected intradermally.

PGEs irritate the mucous membranes of the eyes and respiratory passages. These effects are generally not as 14

immediate or intense as those caused by bradykinin or

histamine .

PGs, notably PGE^, inhibit the basal rate of lipolysis

from adipose tissue in vitro and also lipolysis stimulated

by exposure to catecholamines or other lipolytic hormones.

Such effects have been noted in vivo in various species j

including man. Low doses of PGE^ in man stimulate

lipolysis, presumably by an indirect effect mediated by 44 sympathetic stimulation

It has been suggested that the anti-inflamatory action of aspirin and certain other drugs may be attributed to their ability to block the biosynthesis of PGs^^* If this hypothesis is correct, then PGs may play a fundamental role not only in normal physiological functions but also in certain pathological conditions.

The mechanism of action of PGs is not completely under stood. They affect nearly all physiological processes. To place them in their proper perspective in mammalian physiology various investigators propsed that PGs act as

"autocoids"^^» "modulators"^^j"intracellular messengers"^®, and hormones.

One definition of a "classical hormone" is some substance that on stimulation of a gland or organ is released into the general circulation to act on distant tissues. It is not easy to rule out the possibility that

PGs act as intracellular messengers. Since most cells 15

synthesize PCs in response to hormones and other stimuli,

the newly synthesized PCs could function inside cells as 2+ regulators of cyclic nucleotide and Ca levels, or by 48 directly affecting enzyme activity . In the adipocyte,

PGEs contribute to the regulation of lipolysis by negative

feedback inhibition of 3',5'-cyclic adenosine monophosphate

(c-AMP), but the adipocyte may be an isolated example*^»

Calcium ion is a necessary component of many of the biological actions of c-AMP, and it is veil known that PCs 2+ affect Ca turnover in various systems by stimulating its release from subcellular binding sites^^’^^* In platelets, 2+ added Ca counteracts the inhibition of PGE^ on aggrega tion • It is possible that in some cases, PGs act as intracellular messengers by altering c-AMP levels via 2+ intracellular regulation of Ca transport.

In contrast to this postulation, Bito^^ has proposed that PGs are not intracellular messengers. He argues that

PG synthetase enzyme complex is embeded in cellular membranes in such a manner as to only release newly synthesized PGs into blood or extracellular fluid. There fore, PGs would not accumulate inside cells unless they are actively transported by specific carrier systems, such as are present in the ovaries and major metabolizing organs.

Thus, Bito suggested that PGs act only at cell membrane surfaces as local extracellular hormones. Since PG biosynthesis can be virtually abolished by nonlethal doses 16

of aspirin and indomethacin, their formation can not be

essential for the continued existence of the organism^^.

This fact, and most of what is known about PGs, is in

accordance with the concept that these substances are

secreted into extracellular space, or the local circulation,

to affect nearby cells.

Progress in the field of c-AMP research in the last 10

years has led to the development of the concept that c-AMP

is a "second messenger", that is, the agent of intracellular

information transfer between extracellular hormones and

intracellular enzymes • A large number of tissues respond

to their relevant hormones with increased intracellular

levels of c-AMP. c-AMP is produced by a hormone-sensitive

enzyme, adenylate cyclase, which resides in the plasma membrane and catalyzes formation of c-AMP from adenosine

triphosphate (ATP)^^~^^ (Figure 3). According to the second messenger concept, basal levels of a cellular response are

due to a steady state output of c-AMP, and increases or

decreases in cellular c-AMP concentrations. 17

5'GMP

PDE

Cyclic GMP

PG Contraction 2+ Ca

ATP 5 'AMP Relaxation

PDE

Cyclic AMP

Abbreviations: PDE, phosphorous diesterase; GTP, guanosine triphosphate; ATP, adenosine triphosphate; AC, adenylate cyclase; R, regulatory protein.

Figure. 3 18

In the early 1970s, It was proposed that PG action s are

mediated by Except for the adipocyte, wherein

PGEs lower c-AMP levels in the stimulated cell, PGEs

generally increase intracellular levels of the cyclic

nucleotide in a dose-dependent manner.

Specific binding proteins have been shown to exist for

PGEs in mouse ovary, lipocyte, rat stomach, thyroid, and

other tissues^^’ • Protein receptors have sulfhydryl 19 groups and are located on the cell membrane . The receptors

appear to be specific for PGs and are likely associated with

adenylate cyclase.

PGF analogues only stimulate the synthesis of c-AMP at

high concentrations. At such concentrations PGE receptors

are not distinguishable from PGF receptors. The inability

of PGF2 q; to alter intracellular c-AMP levels provided

evidence that this cyclic nucleotide did not act alone as a

"second messenger" of hormone activity^^*

3',5'-Cyclic guanosine monophosphate (c-GMP) was found to promote cellular events in opposite fashion to c-AMP.

Altered intracellular concentrations of c-GMP are frequently associated with agents that produce effects opposite to c-

AMP. Rather than unidirectional control by c-AMP, Goldberg

5 8 and his associates have suggested that many biological systems are subject to bidirectional control by a balance of the opposing actions of c-AMP and c-GMP. Goldberg proposed the term "Yin-Yang" to describe the phenomenon. 19

Two kinds of bidIrectionally controlled systems are

proposed: A-type systems which are facilitated by c-AMP and

Inhibited by c-GMP, and B-types promoted by c-GMP and

suppresed by c-AMP^^’^^» Cardiac muscle contraction is one

example of an A-type. Contraction of vascular and uterine

smooth muscles, cell proliferation and leukotaxis are B-type 59 systems • B-type systems appear to be more common: release

of lysosomal enzymes from stimulated polymorphonuclear

leukocytes (PMNs) is enhanced by c-GMP and inhibited by c-

AMP*^ •

It was found that 10 ^ M PGFgQ, caused a .four— fold

increase in c-GMP levels in isolated rat uterus preparations

within 43 seconds^^* Oxytocin induced a rapid increase in

c-GMP levels in this tissue, with no further effect upon c-

6 2 AMP levels • Therefore, c-GMP was proposed to be related

to PGF activity and c-AMP related to PGE activity^^» go Recently, studies utilizing isolated bovine and canine veins have shown that the c-GMP/c-AMP ratio increases under the influence of PGF2 q, , whereas the reverse situation

is observed for PGE2 * Since PGï'2q: causes contraction and

PGE2 causes relaxation in this tissue, this finding is consistent with the concept that the opposing actions of E and F prostaglandins are expressed at the cyclic nucleotide level.

PGs are also thought to mediate the activity of luteinizing hormone (LH). Kuehl^^ has suggested that events 20

initiated by LH in promoting ovarian steroidogenesis

involves intermediates in the Scheme shown in Figure 4. In

this Scheme, LH causes the release of PGs which in turn

cause an Increase in the concentration of c-AMP. c-AMP

subsequently activates a protein kinase by binding to and

removing a regulator protein R from the actual catalytic

protein C. This protein kinase catalyzes the enzymatic

production of a phosphorylated protein which is essential

for promoting the synthesis of progestrone.

ATP a LH ^ PGE2 ^ c-AMP ^ ^ Progestrone

step c ATP C-AMP+ RC ^ _ R-C-AMP+ C ^ p-Pr f „ Progestr one

Abbreviations: L H , leuteinizing hormone; ATP, adenosine tri phosphate; R , regulatory subunit; C, cyclic AMP-dependent kinase; P-Protein, phosphorylated protein; c-AMP, cyclic-

AMP.

Figure. 4 Chapter III

PROSTAGLANDIN ANTAGONISTS

PGs seem to be Involved in an ever-increasing number of fundamental responses of cells and organs. Therefore, it is important to have specific PG antagonists to facilitate experimental differentiation and analysis of the roles of endogenous PGs. Furthermore, PG antagonists may be useful therapeutic agents in the treatment of a variety of conditions. The term "prostaglandin antagonist" is used here to denote a compound which selectively antagonizes the actions of PGs at the receptor and not compounds such as aspirin which depress the formation of PGs by inhibiting PG synthetase.

One drug can antagonize the effects of another by four separate and distinct mechanisms: Pharmacological, physiological, physical-chemical and biochemical. A pharmacological antagonist should be specific, potent, reversible and possess no agonist activity. Physiological antagonism takes place when two drugs act at different sites by different mechanisms to produce opposite effects that counterbalance one another. Thus, histamine and slow- reacting substance of anaphylaxis (SRS-A) cause constriction of the bronchioles, impairing normal breathing in asthmatic

- 21 - 22

patients; theophylline and isoproterenol are physiological

antagonists since they produce active dilation of these

respiratory airways by mechanisms not involving PG receptor

interaction. Such compounds are terminators of an acute

asthmatic attack.

Physical-chemical antagonism results when one drug

neutralizes or inactivates another owing to physical or

chemical interaction. Administration of chelating agents or

activated charcoal in the emergency treatment of acute

poisoning are common clinical applications of physical-

chemical antagonism.

Biochemical antagonism occurs when one drug indirectly

decreases the amount of another drug at its site of action.

A drug that decreases the rate at which another drug is

transported across a cell memberane, acts as a biochemical

antagonist. Many drugs have been shown to stimulate drug metabolizing enzyme systems in man or animals. This may

result in one drug activating the metabolism of another drug

that is being given at the same time. The result is a

shortening of its biological half life and a reduction in

its therapeutic effects. For example, exposure of rodents to pesticides such as DDT shortens the duration of action of the hypnotic drug hexobarbital.

One approach used to design antagonists includes synthesis of analogues of the agonist. Certain analogues may have high affinity for a receptor, but due to structural 23

modifications have no intrinsic activity. Such compounds

should act as competitive antagonists. Fried and associ-

ates^*'*^ found that numerous analogues of PGs with oxygen

in place of the methylene group in the 7-posltion of the

molecule specifically inhibited smooth muscle stimulating

properties of PGs. They found that racemic

7-oxoprostaglandin g, had smooth muscle stimulating

properties, but simpler members of the series were devoid of

such activity.

Various analogues (without oxygen functions at

positions 9, 11 and 15) were prepared (Compounds 23).

Many homologues including those having four or six-membered

rings were also synthesized. The degree of hydroxylation appears to be important for determining whether such compounds will behave as agonists or antagonists. Compounds with an oxygen function (hydroxyl or carbonyl) at C-9 and

C-11 were agonists, whether or not there also was a C-15

hydroxyl group (Compounds 2 _ ^ , 2 ^ ) . Compounds with no oxygen functions at C-9 and C-11 and with a C-15 hydroxyl group

(Compounds 2 2 . ) have either PGE^ agonist or antagonist activity. Compounds devoid of an oxygen function at C-9,11 and 15 (Compounds 22, 23) were found to be pure antagonists

(20 ng/ml, 50% inhibition) in the guinea-pig ileum, rabbit jejunum and gerbil colon^^«

The most specific antagonist found by Fried and his

associates'^ was 7-oxo-13-prostynoic acid ( 2 _ ! B ) which has 24

been separated into its optical isomers. The activity for

the two diasteromers has not been reported. Most compounds

(22, 23, 26 and 2 J _ ) were tested and found to produce 50%

inhibition of PGEj-induced contractions on the isolated

guinea-pig ileum and gerbil colon^^ at concentrations of

10-25 and 4-11 pg/ml, respectively.

( C H 2 h

22 23

els and trans

n- 1 , 2

Pure Antagonists

H OH H OH

24 25

Pure Agonists 25

H OH 26 els and trans

Both Agonists and Antagonists

These compounds (32 pg/ml) were found to produce little or no inhibition of contractions induced by AcCh or

histamine on the guinea-pig ileum. Only compounds 21 and 2 T _

(with an acetylenic linkage in the 13,14-position) were specific antagonists for PGE^ on the gerbil colon. The six-membered ring analogues of 7-oxoprostanoic acid caused a shift in 2- and 3-point PGE^ dose-response curves to the right suggesting competitive antagonism. 26

7-0xo-13-prostynolc acid (7-OPyA; 2 3 _ ) and its 15-hydroxy

six-membered ring analogue 2 J _ were also found to inhibit

PGF^Q -induced contractions at concentrations of 10 and 41

IJg/ml, respectively. The most potent analogue, 7-OPyA has become the prototype PG antagonist of this series.

Flack^® tested fifteen of the 7-oxoprostaglandin analogues synthesized by Fried and his associates at a single concentration of 10 pg/ml on the isolated gerbil colon. 7-OPyA produced nearly 100% inhibition of

PGE j^-induced contractions with little or no inhibition of contractions induced by AcCh. Apparently, 7-OPyA was the only selective PG antagonist of the group. However, this compound was nonselective on the guinea-pig ileum and rabbit jejunum. Bennett and Posner^* reported that 7-OPyA was a nonselective PG antagonist on the guinea-pig ileum and colon

(2 -8 x10 ^M) and human stomach , ileum and colon

(5 . 5xlO~'^M-3xlO~^M). Vesin and Herbon^^ observed that 10 and 30 pM concentrations of 7-OPyA inhibited PGE^- induced contractions of the isolated rat uterus by 50 and 100%, respectively. Contractions induced by either oxytocin or carbamoylcholine were not inhibited by 45 pM 7-OPyA. 7-OPyA did not inhibit PGE^-induced increases in c-AMP levels in this tissue, indicating that inhibition of uterine contractions is dissociated from adenylate cyclase inhibition. However, Kuehl and his associates demonstrat ed that 7-OPyA (50-75 pg/ml) blocked the stimulatory effect 27

of PGEj, PGE2 and LH on c-AMP levels in the mouse ovary.

The antagonism was competitive in each case and inhibitor

constants were similar against each of the PG and LH. It was concluded that PGs are involved in stimulation of c-AMP levels induced by LH. 7-OPyA (400 pg/ml) was found to block

PG and LH stimulation of c-AMP formation in bovine luteal cells*^.

c-AMP formation produced by PGs and thyroid stimulating hormone (TSH) in isolated bovine thyroid cells and canine thyroid slices^^"^^ was inhibited by 7-OPyA (10~^M). Ozer and Sharp^^ reported that 7-OPyA (50 pg/ml) reversed PG inhibition of antidiuretic hormone stimulated c-AMP production in isolated preparations of toad bladder. In contrast, Marumo and Edelman^^ demonstrated the PG-like activity of 7-OPyA (10”^M) in hamster kidney homogenates.

7 8 7-OPyA and its 15-hydroxy analogue were found to bind to a lipocyte PG receptor and displace PGE^ from the receptor as would be expected for a competitive antagonist 79 although a high concentration was required. Rao found that 7-OPyA (160 pg/ml) inhibited {^H}-PGE^ binding to human chorionic gonadotropin in cell membranes of bovine corpora lutea. Furthermore, 7-OPyA (10 pg/ml) competed with PGE^ 80 for specific PG binding sites from rat forestomach

Interestingly, 7-OPyA at concentrations of 85 pg/ml also inhibited the enzymatic degradation of PC by

15-hydroxyprostaglandin dehydrogenase (PGDH) purified from 28 O 1 swine lung . The reaction is inhibited in both the reverse 0 2 and forward directions (15-keto PG < ^ 15-hydroxy PG).

7-OPyA was also found to antagonize c-AMP dependent protein kinase^^ and the synthesis of rat ovary proteins®^ ( 1 0 0 pg/ ml). Also, at 15 pM 7-OPyA stimulated ATPase from platel ets, mitochondria and erythrocytes®^* 85 Various dibenzoxazepine derivatives have been shown to specifically inhibit certain PG actions. Competitive PG antagonism was observed on isolated smooth muscle preparations. The prototype compound designated as SC-19220

(29a) was first reported to be a specific PGE2 antagonist on the isolated guinea-pig ileum at concentrations of 1-50 pg/ ml. No significant antagonism of AcCh or bradykinin was observed. Concentrations of 2.5-10 pg/ml shifted PGE2 cumulative dose-response curves to the right in parallel with control curves, indicating competitive antagonism.

Antagonism of PGE2 and PGF2Q. by SC-19220 on the isolated

6 8 69 87 guinea-pig ileum * * , gerbil colon and rat stomach strips®® was observed at concentrations between 5x10”^ and

5xlO"®M. 29

NH — NH—R

(29)

(a) SC-19220 (d) SC-25038

R*=-COCH- R=-C0-(CH2)^CH3

(b) SC-18637 (e) SC-25324

-COCHgCgH^ -C0-(CH2)6CH3

Dibenzoxazepine Derivatives

SC-19220 (5xlO~^ to 10”^M) has been found to inhibit

contractions induced by arachidonic acid, PGE2 and PGF2 q, on

88 89 isolated rat stomach strips * and rat gastric fundus 9 0 muscle • An inhibitory shift in the PGE2 —dose response

91 curve on the isolated sphincter pupillae muscle was

demonstrated with SC-19220 concentrations of 5x10 ^ - 10

Sanner 92 reported that SC-19220, administered

intraperitoneally to mice inhibited diarrhea produced by 30

intraperltoneal administration of PGE2 . When the compound

was suspended with the aid of 0 .1% or 1 % polysorbate 80, the

antidiarrheal potency was increased ten-fold (from an ED^q

of 170 mg/kg to 17 mg/kg). The polysorbate solution

produced no inhibition of diarrhea by Itself, and did not

affect the activity in vitro of the antagonist. 93 Perfusion of the rabbit ear with SC-19220 (3 pg/ml)

selectively reduced the algesic effect of bradykinin but not

that of AcCh. When PG biosynthesis was inhibited by

indomethacin, SC-19220 no longer reduced the bradykinin

effect. However, SC-19220 antagonized the pain-enhancing

action of additionally administered PGE^. It was concluded

that SC-19220 acts by antagonizing the pain-enhancing action

of endogenously released PGs.

SC-19220 (Ixio”^ to lxlO“^M) antagonized the inhibition

of epinephrine stimulated lipolysis by PGE2 in rat

epididymal fat pads*^'*^. SC-19220 had no effect in the

absence of PGE2 " However, Iliano and Cuatrecasas^^ found

that SC-19220 (35 pM) potentiated epinephrine-stimulated

lipolysis in the absence of PGE2 *

With increasing side chain length in dibenzoxazepine

derivatives (Compounds 2 ^ c^, ^ and e^) antiprostaglandin potency on the isolated guinea-pig ileum also increased, but the specificity decreased*^» Alkyl chain branching in the alpha position (29 f) caused a loss in both antiprostaglandin potency and specificity. Phenacetyl derivative SC-18637 31

(29 h ) was more potent against PGE2 on the isolated guinea-

pig ileum than was SC-19220, but SC-18637 was less specific against the PGs.

Polyphloretin phosphate (PPP; ^ ) , a polyanionic polyester of phloretin and phosphoric acid, was synthesized 98 by Diczfaluzy and associates' These polyesters are potent inhibitors of many enzymes including hyaluronidase, alkaline 9 9 phosphatase, and urease. Beitch and Eakins used PPP (10 mg/ml) to stabilize the blood-aqueous barrier in the rabbit eye in a study of the mechanism of intraocular action of PGs on intraocular pressure. They found that the PGs, under these conditions, no longer produced the characteristic increased permeability of the blood-aqueous barrier and did not raise intraocular pressure.

The first report that PPP (5-10 pg/ml) is a selective antagonist of certain smooth muscle actions of PGs came from

Eakins and Karim^^^ using the isolsted gerbil colon.

OH 0 II

T h

30 32

It has been reported that PPP exhibits a reversible

selective inhibition of the contractile responses produced

by both E- and F-type PGs on guinea-pig longitudinal and

circular colonic muscle^* (50-300 pg/ml), gerbil

colon^®®*^®^ (5-10 pg/ml), and rabbit jejunum^®^ (2.5-30

pg/ml). On the isolated gerbil colon, the actions of PGF2

were more readily antagonized by PPP (20 pg/ml) than those

of PGE2 * Contractions induced by ÂcChj bradykinin,

angiotensin and 5-hydroxytryptamine (5-HT) were not affected

102 by concentrations of PPP up to 20 uM • However, PPP

(2 0 - 1 0 0 pg/ml) was found to cause small dose-dependent

contractions of the rat fundus strip^^ and did not antagon

ize responses to PGE2 , PGF2 q- or AcCh.

Different molecular weight fractions of PPP were

separated by chromatography and assayed as PG antagonists

and hyaluronidase inhibitors. The PG antagonist activity

was found only in the low molecular weight fractions which

had little or no ability to inhibit hyaluronidase. The high molecular weight fractions possessed enzyme inhibitory

properties but were not PG-antagonists^^^ * • It has been

found that the most active moieties in PPP were monomers.

Di-4-phloretin phosphate (31), a synthetic compound, was

found to be approximately ten times as potent as PPP^^^« 33

0 II HO — P — 0 No

- ^ 2 31

Contractions induced by PGE2 and PGF2 Q, in isolated

human intestinal muscle preparations^^ and the small and

large intestine of the human fetus^^^ were selectively

antagonized by PPP at concentrations of 100-1,200 and 80-160 pg/ml, respectively. PPP (10-40 pg/ml) antagonized

contractions of the isolated human bronchi^^^lnduced by PGF 2 a ,

caused relaxation of the bronchi which may be related to antagonism of endogenous PGs and antagonized contractions induced by PGE2 in preparations of human umbilical ^ 108,109 arteries

PPP (2.5-30 pg/ml) inhibited contractions of the isolated rabbit uterus^^^* induced by PGEg and PGF2 # * but did not inhibit those produced by either AcCh or epinephrine. PPP (40-60 pg/ml) blocked spontaneous activity induced by PGF2 Q; on uterine strips^^^ taken from normal rats, although it did not affect spontaneous motility and contractions in preparations taken from ovarectomized 34

animals. PPP was found to completely inhibit contractions

induced by PGE^ and " ^ 2 0, » whereas contractions induced by

oxytocin were unaffected^^• Although PPP antagonized

uterine contractions induced by both PGs, it did not affect

the ability to raise c-AMP levels, suggesting that

interaction with adenylate cyclase is not a prerequiste for

the PGs to produce contractions in uterine smooth muscle.

PPP (100-200 mg/Kg; i.v.) was found to prevent the fall

in blood pressure produced by PGF2a , but not that produced

by either PGE2 or AcCh. In the anaesthetised

PPP (200 mg/Kg; i.v.) produced an initial, transient

stimulation of intestinal motility and a fall in blood

pressure. PPP blocked all the effects of PGF2# measured,

namely, an increase in airway pressure, stimulation of

intestinal motility and a fall in blood pressure. However,

PPP only inhibited the effects of PGE2 on intestinal

motility; it did not inhibit the fall in blood or airway

pressure. Therefore, PPP only antagonized some of the PG

actions studied, and in addition enhanced the effects of

PGE2 on intestinal motility prior to blockade^^^» In 17

rats, no fall in blood pressure was seen using up to 640 mg/Kg i.v., and this indicated species differences in

responses to PPP^^^* In five dogs, PPP (lOOmg/Kg; i.v.)

reversed the pressor effect of PGF2 Q, affording a prolonged 113 depressor response • As before, the depressor action of

PGE2 was not inhibited, but was enhanced. 35

Diarrhea produced in mice by intraperitoneal injections

of PGE2 ^was inhibited by PPP (50-200 mg/Kg; i.v.) given

15 min prior to P G . In man, PPP (2 g; orally) did not

prevent diarrhea produced by PGE2 or PGF2q;^ ^ ^ ^ • However,

large doses (4 g) were found to induce diarrhea.

PPP (1.3 pM) was found to be a potent competitive

inhibitor of the PG binding site on PGDH prepared from swine O 1 lung • PPP inhibited pulmonary metabolism of PGs in

isolated perfused guinea-pig lungs^^^, exhibiting an ID^^ of

2.15 and 1.55 pg/ml against PGE2 and PGF2 metabolism^

respectively. At these low concentrations of PPP, no

antagonism of the effects of PGs were seen. PPP (40 mg/Kg;

i.v.) protected guinea-pigs against anaphylactic convulsions

following exposure to antigen aerosols and partially against

convulsions produced by histamine, but it was ineffective against those induced by carbachol^^^ •

PPP (5 pg/ml) inhibited PGE2 and TSH stimulation of adenylate cyclase on isolated bovine thyroid cells, but did not alter basal adenylate cyclase activity in these experiments^^» PPP (5 mg/ml) inhibited PGE^ and LH — stimulated lactic acid production in prepubertal rat 118 ovaries and reversed PGE^ inhibition of osmotic water flow in toad bladders^* at a concentration of 40 pg/ml.

Studies by Kuehl and coworkers^^^ using PPP, indicated that this polymer ( 2 0 0 pg/ml) had no effect upon

PGE^-stimulated c-AMP formation in mouse or rabbit 36

myometrium. Rather, PPP was found to act at a site

subsequent to c-AMP formation. This site was identified as

the c-AMP dependent protein kinase. Therefore, PPP is

considered not to be a true PG antagonist in these tissues

since it does not block the primary PGEj^ event. It has been

shown that phloretin (50 pg/ml) itself antagonized PGE^

stimulation of c-AMP in mouse ovaries^^* These results

suggested that direct PG antagonism by PPP may be related to

the differential abilities of tissues to enzymatically

dephosphorylate the compound to the parent phosphate-free

analogue. Therefore, it appears that the effects of PPP

could be due to direct PG antagonism, inhibition of c-AMP

dependent protein kinase and/or inhibition of PGDH.

Sodium p-benzyl-4— {l-oxo-2-(4-chlorobenzy1)-3-phenyl-

propyl} phenylphosphonate (N-0164; 2&) was found to inhibit

PGE2 ~ and PGF2 Q; -induced contractions on isolated

preparations^^^ of gerbil colon (1-3.8 xlO”^M ) , rat stomach

strips and guinea-pig gastrointestinal muscle

(0.19-3.8xlO”^M). The antagonistic effect on rat stomach

strip preparations to PGE2 and PGF2 a hy N-0164 was shown to

be competitive. The antagonism was reversible and the

dose-response curves in the presence of antagonist were parallel to those obtained in the absence of antagonist.

N-0164 (55 mg/Kg; i.p.) prevented diarrhea induced by

PGE2 in mice. However, N-0164 was less effective orally

(105 mg/Kg). N-0164 (1%) also prevented chemically-induced

irritation (croton oil and pyridine-ether) in the mouse ear. 37

C « , ON q '— ' Q

Recently, Allan and Levi^^^ reported that N-0164 (10

and 1 0 0 ng/ml) was found to selectively antagonize the

vasoconstrictor effects of PGF2 # and PGD2 on the coronary

vasculature of the isolated guinea-pig heart. At higher

concentrations (1.0 pg/ml), N-0164 antagonized the coronary vasodilator actions of PGE2 ; however, it did not modify the

coronary vasodilator action of prostacyclin. N-0164 (10-100 ng/ml) also antagonized sinus rate changes produced by either PGD2 or PGF2Q, , whereas at high concentrations (1 pg/ ml) this compound potentiated sinus rate increases produced by either PGE2 or prostacyclin.

Later, N-0164 (20-100 pM) was found to reverse PGE2 inhibition of isoproterenol-induced c-AMP accumulations in

12 2 rat uterus • N-0164, at the same concentration, was a potent c-AMP phosphodiestrase inhibitor in broken cell preparations and potentiated the c-AMP response to 38

isoproterenol in intact tissues. N-0164 produced similar

proportional increases in the c-AMP response to

isoproterenol in the presence and absence of PGE2 « The

authors suggested that the apparent reversal by N-0164 of

the PGE2 effect on the c-AMP response to isoproterenol is

not due to its PG antagonistic action, but to inhibition of

c-AMP phosphodiesterase. N-0164 (4 pM) selectively

inhibited the PGE2 "lnduced contraction of the rat uterusj

while at higher concentrations it also antagonized

carbachol-induced contractions. Therefore, N-0164 has at

least two effects in the rat uterus; PG antagonism and c-AMP

phosphodiestrase inhibition.

It has been found that morphine (0.01 pg/ml) partially 123 inhibited contractions of Isolated guinea-pig ileum

induced by darmstoff, which was later shown to be a mixture

of PGs. Jaques^^^ showed that morphine and the related

substances, etonitazene (0.23 ng/ml) and nalorphine (0.014 pg/ml) are potent inhibitors of contractions induced by PGE^

on the islated guinea-pig ileum.

Sodium meclofenamate (3^) and sodium flufenamate (34)

at concentrations of 20 pg/ml were found to inhibit

contractions induced by histamine, SRS-A, and PGF2 ^ on human 12 5 bronchial muscle in vitro • Resting tone also was decreased. The vasodepressor response of PGF^^ was selectively blocked by intravenous infusion of meclofenamate

12 6 (30 mg/Kg) into rabbits • Recently, sodium meclofenamate 39

(1 pg/ml) was reported to reduce contractions induced by 90 PGE2 » PCI 2 on the isolated rat gastric fundus

COzNa Cl C O ^ N q o o

a C H ^

(33) (34)

Sodium meclofenamate Sodium flufenamate

Non-steroidal anti-inflammatory compounds such as

aspirin (100 and 300 pg/ml) and phenylbutazone (1 and 10

pg/ml) inhibited contractions induced by PGF2 D: the 125 Isolated human bronchial muscle • Indomethacin (10—60

pg/ml) was found to antagonize contractions induced by 5-HT,

bradykinin and PGE2 on the isolated rat uterus and guinea- 1 27 pig ileum • This PG-biosynthetlc blocker was about four

times more potent against PGE2 « On the isolated rat gastric

fundus^^» indomethacin (1 pg/ml) was found to reduce the contractions induced by PGD2 , PGI2 or PGH2 analogues more

than those induced by AcCh.

Patulin (2: ) was reported to block the effects of irin, later shown to be a mixture of PGs, on the isolated hamster 128 colon Patulin was found to be a nonspecific 40 12 9 antagonist since it inhibited contractions produced on

the isolated guinea-pig ileum by histamine, pilocarpine,

5-HT and AcCh.

(35)

Fatulin

Ethanol (500 ml of 10% solution; i.v.) vas found to

130 inhibit uterine motility in pregnant women only when contractions were induced by PGEj^ and PGF2 # , and not when induced by oxytocin. Also, intracarotid infusion of ethanol^^^ (0.08-0.5%) resulted in inhibition of cerebral vasoconstriction produced by PCE^ and PGF 2Q; in dogs or monkeys .

The inhibition of PG biosynthesis by clidanac^^^

(6-chloro-5-cyclohexyl-l-indancarboxylic acid, TAI-284; 36), 41

Its metabolites and some analogues was assessed using 132 various microsomal preparations as an enzyme source.

Clidanac was found to be a very potent inhibitor of PG 133 - synthetase activity , wherein the (+)-lsomer was shown to

be 1000 times more potent than the (-)-isomer.

CO2H

(36)

Clidanac 134 Experiments on rat fundus smooth muscle preparations revealed that verapamil (5-10 pg), a known Ca^^ antagonist, acts as a PG antagonist. This may explain its ability to act as an antiarrythmic agent, in which PGs are believed to be involved.

A analogue (37) with N,N-dimethylamino substitution on C-1^^^ was found to be a potent, specific

PGF zq antagonist with no agonist activity in either the ■1 O £ gerbil colon (3.2 pg/ml) or the isolated canine lobe 137 preparation (3.2 pg/ml). Intravenous infusion (75 or 150 fig/ml) of 2Z. using anesthetized male Wistar rats^^® was shown to shift the dose-response curves for PGF 2 % -induced 42

pulmonary and systemic vascular effects to the right in a

dose-dependent manner. This analogue exhibited no

significant effect on the pulmonary or systemic vascular

responses to norepinephrine or the pulmonary vascular

responses to arachidonic acid. Therefore, the N,N-dimethyl

analogue of PGF 2a selectively antagonized the pulmonary and

systemic responses to PGF2a in the intact rat in a dose-

dependent fashion.

OH CH. CM

OH OH

There are several other substances which have been

found to inhibit one or more actions of the PGs. These 139 include reserpine on the isolated guinea-pig colon >

isoproterenol(l.5xlO~^ umoles) on the rabbit eye^^°, norepinephrine (lxlO~^ g/ml) on the isolated rat stomach^^l,

estradiol on luteolysis^*^, valinomycin (10"^M) on the

release of growth hormone^^^, progesterone (10 mg; s.c.) on rabbit uterine contractions^^^, and emetine (5-15 pg) on the

isolated rat fundus smooth muscle^^^. 43

Unfortunately, known PG antagonists are not potent when

compared to the therapeutically useful antagonists such as

anticholinergics and antihistamines. Little is known about

their therapeutic usefulness. They have been used as

pharmacological tools to further define the actions of

endogenous PGs.

Several possibilities have been suggested for the

therapeutic applicatin of PG antagonists. PGs are thought

to be involved during labor as a natural uterine

stimulant^**" Thus, PG antagonists could be helpful in preventing habitual abortion or premature labor^^^’ .

Aspirin (200 mg/Kg/day) and indomethacin (2 mg/Kg/day) were

found to block PG synthesis in the uterus, reduce motility and prolong parturition in rats^^^*^^^* These drugs also prolonged mid-trimester saline abortions in humans^^^*

None of the primary PG antagonists (7-OPyA, SC-19220 or

PPP) were found to inhibit relaxation of circular gastrointestinal muscle which is induced by PGE2 * Also, none of the antagonists inhibited all the actions of the

PGs. In this dissertation we describe a new approach to PG antagonist development and the pharmacological results obtained. The rationale is based upon a retrospective analysis of the construction of antihistamines and anticholinergic drugs. Chapter IV

STATEMENT OF THE PROBLEM: A RATIONAL APPROACH TO THE DESIGN OF PG ANTAGONISTS.

To formulate the basis for development of a PG

antagonist a retrospective analysis of the development of

cholinergic blocking agents and H-1 antihistamines is

described. Arguments presented for the development of

antihistamines work equally well for the retrospective

development of a cholinergic blocking agent and thus the

histamlne-antihistamine case is only presented here.

H-1 receptor antihistamines have an aliphatic tertiary

amino group, which is proposed to interact with an anionic

site as does the aliphatic primary amino group of histamine^^^• The tertiary amino group in the antihistamine gains significance because of its longer half-life in vivo.

A primary amine would readily undergo oxidatlve-deamination

rendering the compound inactive. To mimic the size of the

tertiary amine as close as possible to that of the primary amine, small alkyl functions provide antihistamines with higher pA£ values. Larger groups bonded to nitrogen afford considerably less selectivity for the histamine receptors and in the case of prenylamine render a classic antihistamine structure the properties of a Ca antagonist.

- 44 - 45

^ N H 2

Histamine (38)

Antihistamine (39)

?, % — C — 0 — C H 2 — C H 2 — N — M e A^e

Acetylcholine (40) O Me

CH-. Me

Anticholinergic (41)

In addition to an electron-rich nitrogen which would be protonated and H 2 O soluble at physiological pH, an antihistamine requires two aryl groups bonded to some function X which replace the imidazole ring of histamine and provides for increased affinity with loss of intrinsic 46

activity. Similarly, replacement of two hydrogen atoms on

the acetyl methyl group of AcCh by aryl functions converts

the agonist into a cholinergic blocking drug.

Therefore, we propose that PG antagonists might be

constructed having an aliphatic carboxyl group of

appropriate pKa rendering the compound anionic and H 2 O

soluble at physiological pH and capable of interaction with

a pharmacological cationic receptor. Aryl funtionality

bonded to groups having a juxtaposition related to the

cyclopentandiol moiety of PGF2Q; when drawn in the "hairpin"

conformation could, if distances were correct for receptor

site binding, provide increased affinity with loss of

intrinsic activity.

Indeed, conformational analysis of PGs by single

crystal X-ray diffraction techniques^^^ indicated that

active PGs adopt a "hairpin" conformation wherein the ce and w

chains aligned roughly parallel. NMR spectral

studies^^®*in aqueous t-BuOH also suggested the "hair

pin" conformation to be important in biologically active

PGs. However, PGF 2Q has little affinity for PGE2 receptors

and the reverse is also true^^^* Possible differences in

receptor affinity may be related to conformational

differences in solution even though these two analogues

differ only in oxidation state at C (9).

Of all the PGs studied by diffraction techniques, only

PGBj^ does not exist in a "hairpin" conformation. PGA^ as 47

well 35 other "hairpin-shaped" PGs are substrates for PGDH

while PGBj is not^^^- PGBj^ was found to exist preferential

ly in an "L-shaped" conformation. It would appear that PGDH

only metabolizes "hairpin-shaped" PGs • Possibly, the L-

shape of PGBj may physically prevent PGB^ from binding to

the PG site on PGDH or PGB^ may bind to the site but not be metabolized. No experiments have been carried out to differentiate these possibilities.

The Hairpin Conformation of PGF 2 q-

The L-Shaped Conformation of PGB^

Figure. 5 48

Normally, removal of one aryl group in an

antihistamine, wherein potent antagonists always have two

such functions, renders the compound considerably less

actlve^^^'^^^" Thus, the pAg value may drop from 8 to 5.

When this is done, stereoselectivity is observed when the

asymmetric carbon is bonded to the amino N of the molecule,

a phenomenon not observed for the more potent dlaryl

substituted antihistamines. In the latter case, difference

in enantiomeric potency are only observed when the

asymmetric center is CX to the dimethylamino function. These

data suggest that the more potent antihistamines containing

two aromatic rings bond outside the histamine receptor to

regions having little enantiomeric selectivity. The less

potent mono aryl antihistamines may reflect asymmetry of the

histamine receptor per se.

Following this analogy it might be anticipated that di aryl substituted antiprostaglandins would have greater potency when compared to monoaryl analogues, but research is required to elucidate the intricicies of such bonding and the influence of enantioselectivity on both affinity and PG receptor selectivity. In view of the apparent interconversion of PG receptors^^^ the ultimate answer to the development of a selective analogues must result from assessment of a variety of compounds in many pharmacological systems. For these reasons we have extended our studies to include the monoaryl as well as diaryl analogues. 49

OH CO2H

(5)

CO 2 H

(42)

CO2H

(1^) R= R'= 0

(43) R= R'= OH 50

A therapeutically useful antagonist should be

constructed using facile synthetic methodology and in good

yields. For these reasons, we have employed the bicyclic

indanone and indanol structure which mimics carbons 4-8 and

12-16 in PGF2q, • Furthermore, the high electron density at

position 6,13 and 14 have a juxtaposition similar to the

appropriate tt cloud of the aromatic ring in the indanone

structure. Furthermore, it is known that the ISO!-hydroxyl 162 group of PGF20! is required for intrinsic activity

Oxidation to the ketone decreases such potency. In

proposing a PG antagonist, the indane hydroxyl group or the

carbonyl in its indanone counterpart have a juxtaposition to

the 15 Ct-hydroxyl group of PGF2Q: relative to the carboxyl

function like a 15Cü-hydroxyl or a carbonyl group of PGF2 q;»

Bonding of the flexible n-Bu or other alkyl groups to the

2-position of the indane ring as well as thep-carboxyethyl function provide both the necessary lipophilicity and flexibility to accomodate a receptor interaction. Thus, this first approximation to antagonist design does not require the absolute fit of a completely rigid molecule.

The two phenolic hydroxyl groups at positions 5 and 6 of the indanone structure mimic C-9 and C-11 of PGF2 q; and serve for bonding aryl functionality in the form of benzyl groups at the appropriate distance from the carboxyl function. The benzyl groups afford a flexibility allowing for bonding to a number of possible sites outside the immediate vicinity of a 51 hypothetical PG receptor. In our PG antagonist structure, we have deleted C-10 of PGF2 # and have rendered C-4 and C-16 a single C at position 2 of the indanone.

CO 2 H

R= Et (44)

R= n-Pr (45)

R= n-Bu (46)

R= n-Pen (4 7 )

R= CH2 CH 2 CO 2H (48)

■0 CO 2

R= Et (49)

R= n-Pr (50)

R= n-Bu (51)

R= n-Pen (52) Chapter V

RESULTS AND DISCUSSION

The syntheses for targets 44-52 are found in Schemes I

and II.

Cyclization of 3-hydroxyphenylpropionic acid (53)

prepared^** from m-hydroxyhenzaldehyde and malonic acid

followed by catalytic hydrogenation of the resulting

cinnamic acid, was accomplished using AlClg (NaCl)/180-200°C

in a procedure described by Bruce and coworkers^• Steam

distillation^** of the mixture provided non-volatile

5-hydroxy-l-indanone (5_4) in 85% yield- Indanone 5 ^ was

isolated from the distillate in 15% yield. Treatment of

with K 2 C0 2 /abs.EtOH/CgH3 CH2 Cl afforded intermediate,

5-benzyloxy-l-indanone (5^) in 87.5% yield. Condensation of

56 with dimethyl carbonate in the presence of NaH afforded

carbomethoxy analogue 5 7 _ in 95% yield.

Previous attempts to prepare 2-alkyl substituted

indanone from 2Z. via the enamine failed owing to poor

enamine yields^. Alkylation of 5_7 using Na/n-alkyl halide

or NaOEt/n-alky 1 halide in EtOH also only afforded poor

yields (5%) of the alkyl derivative^- However, use of NaH/

n-alkyl bromide in freshly distilled THF containing hexamethylphosphoramide (HMPA) afforded 58-61 in 68-82%

- 52 - 53 yield. Hydrolysis and subsequent decarboxylation in acetic acid: cone HCl: H 2 O (2.5: 1: 1) afforded indanones 62-65 in

65-78% yield.

a Scheme I.

OOH +

II OH

COOMe MeO-C-OMe

IL NaH R-Br HÏ1PA

PhCH,0

‘COOMe HOAc

0 62-65

COOMe

0 66-69 R= Et, n-Pr, n-Bu, n-Pen 54

Anion formation using t-BuOK in t-BuOH followed by Michael

addition to methyl acrylate yielded indanone esters 66-69

following chromotography on silicic acid using

toluene : chloroform : ethyl acetate (75:20:5). Alkaline ester

hydrolysis afforded keto acids 44-47 in 60-70% overall yield

from 62-65.

Reduction of the intermediate ester 6^ with NaBH^ in

MeOH, however, did not afford the desired epimeric indanols

7 0 , but rather two tricyclic compounds 71_ (30%) and 7_2 (50%)

which were separated by chromatography on silica gel using

CHCl3 :i-PrOH (96:4) as eluting solvent. The indane C-1

proton resonance signals for T l _ ( 6 5.38) and 7^ ( 6 4.54)

were particularly diagnostic. For 7 2, the additional two

proton resonance signal multiplets (X) bonded to the C a to

1 5 oxygen appeared at 6 3.5-3.7. The { C}-carbonyl resonance 13 signal for 7^ appeared at 172.4 ppm and the equivalent { C}

signal for 7 2 appeared at 63.5 ppm. As expected, the resonance signal chemical shifts for 21. and 7_2 were identical (70.36 ppm). o C O ^ H

(70) 55

Alternatively, reduction of the corresponding keto acid anlons^^® (Na salts of 44-47 ) using NaBH^, E 2O for 4-6 days at room temperature afforded predominantly the respective trans-hydroxy acids (49-52) in 80-95% yield. For

49, lactone 22 likely arising from cyclization of cis- hydroxy acid also was isolated in 15% yield. The indane C-1 proton resonance signal for the trans-hydroxy acids (49-52 ) appeared at 5 4.72-4.75, whereas the equivalent signal in lactone 7_3 again appeared downfield ( 6 5 .39). Comparison of 13 the { C}-C-l resonance signal for ketone 4_^ (208.42 ppm) and alcohol 51 (78.97) confirmed structural assignments.

X= 0, R= n-Bu (71)

X= H 2 » n-Bu (72)

X= 0, R= Et (73) 56

Comparative stereoselective reduction of keto ester 68

with NaBH^-MeOH affording cis-lac tones and ethers ( J J l and

72 ) with reduction of keto acids (44-47 ) using

NaBH^/NaOH/H2 Û affording trans-hydroxy acids (49-52)

deserves comment . For ester NaBH^ reduction is sensitive

to steric approach control^^^’ in MeOH. Solvation of the

carbomethoxy group of 6^ by MeOH provides for increased

steric bulk over the n-Bu group directing hydride attack on

the carbonyl carbon from the n-Bu side. Rationales involving electrostatic shielding^in the case of borohydride reduction of anions are excluded since such rationales should predict stereoselective attack from the side opposite the anion. House and coworkers^^^* assumed that the geometry of the transition state and final product are similar and adequate to predict the major stereochemical course of borohydride reductions providing that the molecule is not conformationally constra ned such that attack is prevented from one side of the carbonyl. However, the high degree of stereoselectivity observed for reduction of 44-4 7 and the opposite stereochemical outcome when compared to reduction of 68 suggests to us that assisted hydride transfer via intramolecular solvation of the borohydride ion^^^ 1s important. Since the magnitude of the non-bonded interaction between the formed hydroxy moiety and cis-alky1 or cis-propionate group is expected to be similar, invoking 169,171 concepts of product development control seems not to be appropriate. 57

5-Benzyloxy-l-lndanone (5^) served as starting material

for preparation of dicarboxylic acid via ester 7_4. Anion

formation followed by Michael addition to methyl acrylate

and alkaline hydrolysis afforded 4^ in 60% overall yield.

Reduction of 4_8 using one molar equivalent of NaBH^ and NaOH

afforded lactone 7_5 in 90% yield. The indane C-1 proton

resonance signal at g 5.4 was consistent with the signal

observed for C-l-H in 7^ and 7 3.

a Scheme II.

0 0 ^ 7± R = R = CH2CH2C02h4e 46 R= R = CH2CH2 CO2 R

a a= t-BuOK, t-BuOH, r.t.; b= CH2®CHC02Me, r.t.; c= NaOH, H 2 O,

MeOH; d, NaBH^, NaOH, B^O, r.t., 4 days. Chapter VI

EXPERIMENTAL SECTION

Melting points were determined on a Thomas Hoover apparatus and are uncorrected. { H-} and { C-} NMR spectra were obtained using Bruker 90 and 80 MHz Instruments, respectively. Infrared spectra were recorded using a Beckman IR 4230 spectrometer. Mass spectra were determined using a Du Pont 491 Instrument. Elemental analyses were obtained from Galbraith Laboratories Inc., Knoxville, TN.

5-Hydroxy-l-ldanone(54). A mixture of anhydrous AlClg

(10.0 g; 0.073 mol) and NaCl (2.0 g; 0.05 mol) was melted In

a beaker by direct beating over a flame while stirring with

a glass rod. 3-Hydroxypbenylproplonlc acld^^* (5^ ; 2.0 g;

0.012 mol) was added at 140°C. The melt temperature rapidly

Increased to 1 8 0 °C and was maintained at 180-200°C for 2

mln. The residue was steam distilled and

7-hydroxy-l-lndanone (5_5) crystallized from the distillate.

5-Hydroxy-l-lndanone ( 5_4 ) remained In the flask and was

filtered. Recrystalllzatlon from MeOH afforded yellow

needles, mp 181—183°C (llt.^^^ mp 184—184.5°C).

5-Benzyloxy-l-lndanone(56). To a mixture of

5-hydroxy-l-lndanone (5^ ; 3.25 g; 0.022mol), and anhydrous

K 2 CO 3 (3.4 g; 0.022 mol) In absolute EtOH (100 mL) benzyl

chloride (3.0 g; 0.022 mol) was added dropwlse with stirring

- 58- 59

under N 2 • The mixture was refluxed over night, filtered and

washed with EtOH. The combined filtrates were concentrated under reduced pressure affording a yellow residue which upon

recrystallization from EtOH afforded 4.9 g (87.5%) of color

less crystals mp 105—106°C (lit.^^^ mp 105.5—106°C).

5-Benzyloxy-2 - carbomethoxy-l-indanone(57 ) . To a slurry of NaH (1.2 g; 0.05 mol; 50% emulsion) in dimethyl carbonate

(10 mL) was added with stirring under Ng a solution of

5-benzyloxy-l-indanone ( ^ 6 ; 4.76 g; 0.02mol) in dimethyl carbonate (70 mL) . The mixture was refluxed for 3 h, after which time the excess NaH was decomposed by dropwise addition of ice cold H 2 O followed by acidification with glacial HOAc. The solution was extracted with Et 2 Û and the

Et20 layer was washed, dried (Na2 S0 ^), filtered and cone. under reduced pressure affording a yellow solid. Recrystal lization from MeOH afforded 5.6 g (95%) of light yellow crystals mp 1 0 2 - 1 0 3 °C; IR (KBr) 1740, 1710 cm"^; {^H-} NMR

(CDClg) 6 6.80-7.70 (m, 8 H, aromatic), 5.1 (s, 2H, benzylic),

3.75 (s, 3H, - C O 2 CH 3 ), 3 .2-3 .8 (m, 3H, ABC pattern of 2H-3 and H—2) .

A n a l . Calcd for Cj^gHji^O^: C, 72.96; H, 5.44. Found:

C, 73.02; H, 5.45.

5-Benzyloxy-2n-butyl-2-carbornetboxy-1-indanone(60) .

To a slurry of NaH (1.2 g; 0.05 mol; 50% emulsion) in 10 mL 60

of THF (freshly distilled from LAIH^) was added with

stirring under N 2 a solution of 5-benzyloxy-2-carbomethoxy-

1-indanone (5_^ ; 5.92 g; 0.02 mol) in THF (60 mL) . The mixture was heated at 60-70°C for 15 min and n— butyl bromide

(3.01 g; 0.022 mol) was added. The mixture was refluxed for

5 h, cooled and 15 mL of HMPA (highly carcinogenic) was added. The clear solution was stirred at room temperature

for 1 h and refluxed overnight. Following concentration under reduced pressure, H 2 O was added and the mixture extracted several times with Et 2 0 . The organic layer was dried (Na2 S0 ^), filtered and concentrated under reduced pressure affording 5.81 g (82.5%) of colorless crystals mp

8 6 -8 8 °C (benzene/pet.ether). IR (KBr) 1750,1710 cm ^ ; {^H-}

NMR (CDCI3 ) 56.8-7.8 ( m, 8H, aromatic), 5.1 (s, 2H, benzyl ic), 3.6 (s, 3H, -CO2 CH3 ), 0.7-2.1 (m, 9H, aliphatic).

Calcd. for the AB part (benzylic) 5 ^ 3.68, 5 g 3.04 with

= 17.48 H z .

Anal. Calcd for C2 2 H 2 4 O 4 ! C , 74.97; H, 6 .8 6 . Found:

C, 74.88; H, 6.91.

5-Benzyloxy-2n-butyl-l-lndanone(64). To a solution of

5-benzyloxy-2n-butyl-2-carbomethoxy-l-indanone (6^ ; 1.72 g;

0.005 mol) in glacial HOAc (10 mL) was added 10 mL of d il

HCl solution. The mixture was refluxed for 5 h, stirred overnight at room temperature, and concentrated under reduced pressure. The residue was dissolved in Et 20 and 61

washed with H 2 O and saturated NaCl solution, dried (Nag^SO^),

filtered and concentrated under reduced pressure affording

1.05 g of solid. Debenzylated product was separated by

chromatography on silicic acid using toluene: CHCI3 ; EtOAc

(75: 25: 5) as the eluting solvent. Benzylated indanone 6^

eluted first and following solvent removal under reduced

pressure was recrystallized from MeOH affording 1.07 g (73%)

of colorless crystals mp 6 6 - 6 8 °C. IR (KBr) 1710 cm ^ ; {^H-}

NMR (CDCI3 ) 6 6.8-7.74 (m, 8H, aromatic), 5.1 (s, 2Hj

benzylic), 2.5-3.2 (m, 3H, ABC pattern of 2H-3 and H-2 ) j

0.81-1.6 (m, 9H, aliphatic).

A n a l . Calcd for 020^22^2" 81.59; H, 7.53. Found;

C, 81.42; H, 7.60.

The debenzylated product, 2n-butyl-5-hydroxy-l—indanone

{0.19 g(12%; MeOH)} exhibited mp 134-136°C. {^H-} NMR

(DMSO-dg) 5 6.6-7.55 (m, 3H, aromatic), 2.3-3.4 (m, 3H, ABC

pattern of 2H-3 and H-2), 0.8-1.55 (m, 9H, aliphatic).

Anal. Calcd for Cj^3 Hj^g0 2 î C , 76.44; H, 7.89. Found:

C, 76.51; H, 7.90.

Methyl 5-Benzyloxy-2n-butyl-l-oxo-2-indanpropionate(68)

and Its Free Acid(46 ) . To a solution of 5-benzyloxy-

2n-butyl-l-indanone (64 ; 2.94 g; 0.01 mol) in t-BuOH (25 mL) was added t-BuOK (0.44 g; 0.004 mol). The mixture was

stirred at room temperature under N2 for 5 h. Methyl 62

acrylate (1.72 g; 0.02 mol) was added and the mixture was

stirred at room temperature overnight, acidified with

glacial HOAc, and concentrated under reduced pressure. The

solid was dissolved in £t 2 Û, washed twice with 1 0 % N a 2 C0 g

solution, dried (Na2 S0 ^) filtered and concentrated under

reduced pressure. The residue was chromatographed on

silicic acid using toluene; CEClg: EtOAc (75: 25: 5) as

eluting solvent. The resulting colorless oil obtained

following solvent removal was hydrolysed using 15% methanol-

ic KOH reflux for 2 h) thereby affording a yellow semi-solid

which was recrystallized from benzene/hexane yielding 2.7 g

(74%) of 46 as white crystals mp 76-78^C. IR (KBr) 1740,

1715 cm"^; NMR (CDCl^) £10.22 (s, IE, COgE), 6.72-7.8

(m, BE, aromatic), 5.12 (s, 2E, benzylic), 0.62-2.4 (m, 13E, aliphatic). Calcd for the AB part (benzylic) £ ^ 3.05, £g

2.84 with J^B *= 17.59 Ez; MS (70 eV), m/e 3 6 6 (m '*');

NMR (CDCI3 ) 208.43 (C=0), 178.48 (CO2H).

A n a l . Calcd for C2 3 H 2 6 O 4 : C, 75.38; H, 7.15. Found:

C, 75.23; E, 7.26.

Sodium Borohydride Reduction of Methyl 5-Benzyloxy-2n- butyl-1-0 X 0 - 2 — indanpropionate(6 8 ). To a solution of methyl

5-benzyloxy-2n-butyl-l-oxo-2-indanpropionate ( 68 ; 1.9 g;

0.005 mol) in MeOH (20 mL) was added with stirring NaBH^

(0.189 g; 0.005 mol) in E 2 O (10 mL). The mixture was stirred for 20 h at room temperature and 10 mL of 1 N NaOH 63

solution was added. The MeOH was removed under reduced

pressure and to the resulting semi-solid was added cone HCl:

H 2O (1:1). The aqueous mixture was extracted with Et 20 and

the organic layer was dried (Na2 S0 ^), filtered and

concentrated under reduced pressure affording an oil (1 . 5 3

g) which was chromatographed on silica gel {CHCI3 :i-PrOH

(96:4)}.

(A)(cis)-5-Benzyloxy-2 n-buty1-1-hydroxy-2-indanpropionic

Acid, S-Lactone(71) 0.52g(30%) was obtained as colorless

crystals mp 1 1 1 -1 1 3 °C following solvent removal under

reduced pressure and recrystallization from

CHCI3 /benzene/hexane. IR (KBr) 1730 cm ^ ; {^H-} NMR (0001^)6

6.78-7.44 (m, 8H, aromatic), 5.38 (s, IH, C-1 H), 5.05 (s,

2H, benzylic), 0.8-2.49 (m, 13H, aliphatic). Calcd for the

AB part (benzylic) {Bruker 300 MHz instrument} 6 ^ 2.97, 6 g

2.88 with = 16.68 Hz; MS (70 eV)m/e, 350(M*); {^^C-} NMR

(CDCI3 ) 172.41 (C=0), 70.36 (C-1 ato 0).

Anal. Calcd for C 2 3 H 2 5 O 3 : C, 78.83; H, 7.48. Found:

C, 79.09; H, 7.70.

(^)(cis)-7-Benzyloxy-4a-n-buty1-2,3,4,4a,5,9b-hexahydro- indeno-{l,2-b}pyran(72) 0.85g(50%) was obtained as colorless crystals mp 9 4 - 9 5 °C following solvent removal under reduced pressure and recrystallization from benzene/CH30K . {^H-}

NMR(CDCl3 ) 66.8-7.44 (m, 8 H, aromatic), 5.04 (s, 2H, 64

benzylic), 4.54 (s, IH, C-1 H), 3.49-3. 69 (m, 2H, -O-CH2 ),

0.87-1.68 (m, IIH, aliphatic). Calcd. for the AB

part( benzylic) 2.74, g g 2.65 with Jy^g = 15.56 Hz; MS (70

eV); m/e 336(M*); {^^C-} NMR (CDCI3 ) 70.36 (C-1 a to 0);

63.52 (-O-CH2 ).

Anal. Calcd for C2 3 H 2 7 O 2 : C,82.22; H, 8.1. Found: C,

82.33 ; H, 8.4.

(trans)-5-Benzyloxy-2n-buty1-1-hydroxy-2-indanpropioni c

Acid(51). A solution of 5-benzyloxy-2n-butyl-l-oxo-

2-indanpropionic acid ( ^ ; 1.1 g; 0.003 mol) in H^O (15 mL)

containing NaOH (0.12 g; 0.003 mol) was added to NaBH^

(0.114 g; 0.003 mol) in 0.2 N NaOH (1 mL). The mixture was

stirred at room temperature for 4 days, slowly acidified

with dil HCl solution and extracted with Et 2 0 . The Et 20

layer was extracted with 10% aqueous NaHCOg solution. The

aqueous layer was acidified with dil HCl solution and

extracted with Et 2 0 . The E t 20 solution was dried (Na2 S0 ^),

filtered and concentrated under reduced pressure.

Recrystallization of the residue from EtOAc/benzene/hexane

afforded 0.92 g (85%) of white crystals mp 145-147°C. IR

(KBr) 3400, 1685 cm'^; {^H-} NMR (CDCl^) Ô 6.77-7.45 (m, 8H,

aromatic), 5.03 (s, 2H, benzylic), 4.72 (s, IH, C-1 H), 2.63

(s, IH, -CO2 H), 0.84-2.46 (m, 13H, aliphatic). Calcd for

the AB part (benzylic) 6 y^ 2.95, 2.43 with Jy^g = 15.89 Hz;

MS (70 eV), m/e 368(M*); {^^C-} NMR (DMSO-dg) 184.24

(-CO2 H), 78.8 (C-1 OH). 65

Ana1. Calcd for 74 . 96 ; H, 7.65. Found:

C, 74.65; H, 7.53.

5-Ben2 yloxy-l-0 X 0 - 2 ,2-lndandlpropionlc Acid (48).

5-Benzyloxy-l-indanone (5^ ; 2.38 g; 0.01 mol) was treated

with t-BuOK (0.56 g; 0.005 mol) in t-BuOH (20 mL) under N 2

with stirring. Methyl acrylate (1.72 g; 0.02 mol) was added

and stirring continued overnight. The mixture was acidified

with glacial HOAc, concentrated under reduced pressure and

diluted with Et 2 0 . The organic layer was washed with H 2 O,

dried (Na2 S0 ^) and conc. under reduced pressure affording a

viscous oil (2.3 g) of dimethyl ester 7_4 which was not

further purified, but hydrolysed to diacid 48 using a 10%

NaOH solution (Me0H:H 2 0 ; 2:8). Diacid was recrystallized

from HeOH/benzene/hexane affording 2.2 g (60%) of colorless

crystals mp 144-145°C. IR (KBr) 3400, 1730, 1700 cm ^ ;

NMR (CDClg) 5 6.93-7.79 (m, 8H, aromatic), 5.14 (s, 2H,

benzylic), 2.94 (s, 2H, benzylic), 1.96-2.3 (m, 8H,

aliphatic); MS (70 eV), m/e 382(N^)-

Anal. Calcd for ^ 22^ 22^ 6 ’ C,69.09; H, 5.80. Found:

C, 69.08; H, 6.01.

(trans)-5-Benzyloxy-l-hydroxy-2 ,2-indandipropionic

Acid, 5 Lactone(75). Using conditions virtually identical to

those employed in the reduction of keto acid 4^, afforded

5-lactone 7_5 mp 1 7 5 -1 7 6 °C ( CHCl^/benzene/hexane) in 90% 66 yield. IR (KBr) 3400, 1740, 1700 cm"^; NMR (CDCl^yS

6.8-7.67 (m, 8 H, aromatic), 5.4 (s, IH, C-1 H), 5.05 (s, 2H, benzylic), 2.95 (s, 2H, benzylic), 1.25-2.48 (m, 8 H, aliphatic); MS (70 eV), m/e 366(M^).

Anal. Calcd for ^22^22^5’ ^ * 72.11; H, 6.05. Found:

C, 71.77; H, 6.18.

5-benzyloxy-2-carbornethoxy-2n-penty1-1-indanone(61).

To a slurry of NaH (1.2 g; 0.05 mol; 50% emulsion) in 10 mL of THF (freshly distilled) from (LiAlH^) was added with stirring under N2 a solution of 5-benzyloxy-2-carbomethoxy-

1-indanone (^7_ ; 5.92 g; 0.02 mol) in THF (60mL). The mixture was heated at 60— 7 0 °C for 15 min. and n-pentyl bromide (3.32 g; 0.022 mol) was added. The reaction mixture was worked up using methodology identical to that described for the preparation of 6 0 . Recrystallization from benzene/ hexane afforded 5.22 g (71%) of colorless crystals mp

48-50°C. IR(KBr) 1745, 1705 cm"^; {^H-} NMR (0001^)6

6 . 89-7 .73 (m, 8 H, aromatic), 5.12 (s, 2H, benzylic), 3.66

(s,3H, -CO2 CH3 ), 0.82- 2.67 (m, IIH, aliphatic). Calcd for the AB part (benzylic) 6^ 3.64, ô g 2.99 with J^g = 17.48 Hz.

Ana 1. Calcd for Cg^Eg^O^: C, 75.38; H, 7.15. Found:

C, 75.55; H, 7.25.

5-Benzyloxy-2n-pentyl~l-indanone(65). To a solution of

5-benzyloxy-2-carbomethoxy-2n-pentyl-1 -indanone (j^ ; 1.83g; 67

0.005 mol) in glacial HOAc (10 mL) was added 10 mL of dil

HCl solution. The reaction mixture was worked up using

methodology identical to that described for the preparation

of 6 ^ , Recrystallization from Me0 H/H 20 afforded 1.0 g (65%)

of colorless crystals mp 69-71°C. IR (KBr) 1700 cm ^ ; {^H-}

NMR (0 0 0 1 3 ) 6 6.63-7.50 (m, 8H, aromatic), 5.13 (s, 2h;

benzylic), 2.53-3.27 (m, 3H, ABO pattern of 2H-3 and H-2 ),

0.90- 1.96 (m, IIH, aliphatic).

Anal. Oalcd for C2 1 H 2 4 O 2 : 0,81.79; H, 7.84. Found :

0,81.84; 8.00.

Methyl 5-Benzyloxy-1-oxo-2n-pentyl-2-indanpropionate

(69) and Its Free A c i d (47 ). To a solution of

5-benzyloxy-2n-pentyl-l-indanone (^5^ ; 3.08 g; 0.01 mol) in

t-BuOH (25 mL) was added t-BuOK (0.44 g; 0.004 mol). The mixture was stirred at room temperature under N2 for 5h.

Methyl acrylate (1.72 g; 0.02 mol) was added and stirring

continued overnight. The reaction mixture was worked up using methodology identical to that described for 6 8 .

Recrystallization from MeOH afforded white needles mp

7 1 -7 3 °C. IR (KBr) 1750, 1710 cm“^; {^H-} NMR (0001^)0

6.93-7 . 72 (m, 8 H, aromatic), 5.14 (s, 2H, benzylic), 3.61

(s j 3H, -CO2 CH 3 ), 0.83-2.32 (m, 15H, aliphatic). Calcd for

the AB part (benzylic) 6 ^ 3.03, 6 g 2.83 with = 17.92 Hz.

Ana1. Calcd for C, 76.14; H,7.66. Found :

C, 76.39; H, 7.91. 68

Hydrolysis of ester 6^ afforded a yellow semi-solid

which was recrystallized from CHCI3 /benzene/hexane to give

2.5 g (65%) of white crystals mp 69-71°C. IR (KBr) 3400,

1730, 1690 cm“^; NMR (CDCI3 ) 6 6.91- 7.71 (m, 8 H, aromatic), 5.12 (s, 2H, benzylic), 0.81- 2.32 (m, 15H, aliphatic). Calcd for the AB part (benzylic) 6 ^ 3.03, 6 g

2.82 with = 17.8 Hz; MS (70 eV), 380(M*).

A n a l . Calcd for C 2 4 H 2 gO^: C, 75.76; H, 7.41. Found ;

C, 75.50; H 7.25.

( trans)-5-Benzyloxy-1 —hydroxy-2n-pentyl-2-indanpropionic

Acid (5^). To a solution of NaBH^ (0.114 g; 0.003 mol) in

0.2 N NaOH (1 mL) was added a solution of 5-benzyloxy-1-cxo-

2n-pentyl-2-indanpropionic acid (42 ; 1.14 g; 0.003 mol) in

H 2 O (15 ml) containing NaOH (0.12 g; 0.003 mol). The mixture was stirred at room temperature for 6 days. The reaction mixture was worked up using methodology identical to that described for the preparation of 51 .

Recrystallization from CHCI 3 /benzene/hexane afforded 0.97 g

(85%) of white crystals mp 1 4 1 — 1 4 2 °C. IR (KBr) 3400, 1680 cm ^; {^H-} NMR (CDCl^) 6 6.78-7.72 (m, 8 H, aromatic), 5.04

(s, 2H,benzylic), 4.72 (s, IH, C-1 H), 0.82-2.45 (m, 15H, aliphatic), calcd for the AB part (benzylic) 2.96, f g

2.44 with = 16.21 Hz; MS (70 eV) 3 8 2 (m ‘‘’).

A n a l . Calcd for C 2 4 H 3 q O ^ : C,75.38; H, 7.90. Found :

C, 75.35; H, 8.04. 69

5-B enzyloxy-2-carbomethoxy-2n-propy1- l-indanone(59) .

To a slurry of NaH (1.2 g; 0.05 mol; 50% emulsion) in 10 mL

of THF (freshly distilled) was added with stirring under N 2

a solution of 5-benzyloxy-2-carbomethoxy-l-indanone (52^ ;

5.9 g; 0.02 mol) in THF (60 mL). The mixture was heated at

60-70°C for 15 min and n-propyl bromide (2.7 g; 0.022 mol)

was added. The reaction mixture was worked up using methodology identical to that described for the preparation

of 60. Recrystallization from benzene/hexane afforded 5.7 g

(75%) of light yellow crystals mp 72-74°C. IR (KBr)1735,

1705 cm ^NMR (CDCl^) 6 6.97-7.76 (m, 8H, aromatic),

5.15 (s, 2H, benzylic), 3.7 (s, 3H, -CO2 CH3 ), 0.8-2.07 (m,

7H, aliphatic). Calcd for the AB part (benzylic) 5^ 3.65,

6 g 3.01 with - 17.48 Hz; MS (70 eV), m/e 338(M*).

A n a l . Calcd for C2 7 H 2 2 O 4 : C , 74.53; H, 6.55. Found:

C, 74.55; H, 6.47.

5-Benzyloxy-2n-propyl-l— indanone(63). To a solution of

5-benzyloxy-2-carbomethoxy-2n-propyl-l-indanone (22. » 1*69 g; 0.005 mol) in glacial HOAc (10 mL) was added 10 mL of dil HCl solution. The reaction mixture was worked up using methodology identical to that described for the preparation of 6^* Recrystallization from MeOE/H20 afforded 1.09 g

(78%) of colorless crystals mp 5 5 -5 7 °C. IR (KB) 1700cm ^;

{^H-} NMR (€0 0 1 3 ) 6 6 .9 5 - 7 . 7 5 (m, 8H, aromatic), 5.14 (s, 2H, benzylic), 2.66-3. 33 (m, 3H, ABC pattern of 2H-3 and H-2),

0.88-1.98 (m, 7H, aliphatic); MS (70 eV), m/e 280(M+), 70

A nal. Calcd for Cj^gH2o02’ C,81.38; H,7.18. Found:

C, 81,44; H, 7,37.

Methyl 5-Ben2yloxy-l-oxo-2n-propy1-2-IndanpropIonate

(67 ) and Its Free Acld(45 ). To a solution of

5-benzyloxy-2n-propyl-l-indanone ; 2.8 g; 0.01 mol) in

t-BuOH (25 mL) was added t-BuOK (0.44 g; 0.004 mol). The

reaction mixture was stirred at room temperature under N 2

for 5 h. Methyl acrylate (1.72 g; 0.02 mol) was added and

stirring continued overnight. The reaction mixture was

worked up using methodology identical to that described for

68 . Ester 7^ was not further purified, but hydrolyzed to

acid 4^ using 10% NaOH solution (MeOH: H 2 O; 2: 8 ).

Recrystallization from benzene/hexane afforded 2.46 g (70%)

of colorless crystals mp 1 2 2 -1 2 4 °C. IR (KBr) 3400, 1740,

1705 cm ^; {^H-} NMR (CDClg)g 6.94-7.73 (m, 8H, aromatic),

5.14 (s, 2H, benzylic), 0.85-2.3 (m, IIH, aliphatic). Calcd

for the AB part (benzylic) g^ 3.06,g g 2.85 with = 17.46

Hz; MS (70 eV), m/e 3 5 2 (M*).

Ana 1. Calcd for 022^24^4" 74.97; H, 6 .8 6 . Found:

C, 75.04; H, 6.87.

( trans)-5-Benzyloxy-1-hydroxy-2n-propy1-2-indanpropionic

Acid (5_0). To a solution of NaBH^ (o.l4 g; 0.003 mol) in

0.2 N NaOH (1 mL) was added a solution of 5-benzyloxy-1-0 x 0 -

2n-propy 1-2-indanpropionic acid ( ^ ; 1.06 g; 0.003 mol) in 71

H 2 O (15 mL) containing NaOH (0.12 g; 0.003 mol). The

reaction mixture was worked up using methodology identical

to that described for the preparation of 51.

Recrystallization from MeOH/benzene/hexane afforded 1.0 g

(95%) of white crystals mp 125-127^0. IR (KBr) 3500, 1700

cm ^NMR (CDCl^) 5 6 . 8 (m, BE, aromatic), 5.05 (s, 2H,

benzylic), 4.75 (s, IE, C-1 E), 0.93- 2.47 (m, H E ,

aliphatic). Calcd for the AB part (benzylic) 6 ^ 2.99, 6 g

2.44 with J^g - 16.2 Ez; MS (70 eV), m/e 3 5 4 (m ‘‘‘).

A n a l . Calcd for 74.56; E, 7.39. Found:

C, 74.47; E, 7.45.

5-Benzyloxy-2-carhomethoxy-2-ethy1-1-indanone(58) . To

a solution of NaE (1.2 g; 0.05 mol; 50% emulsion) in 10 mL

of TEF (freshly distilled) was added with stirring under N 2

a solution of 5-benzyloxy-2-carbomethoxy-l-indanone (22. • »

5.9 g; 0.02 mol) in TEF (60 mL). The mixture was heated at

60-70*^C for 15 min and ethyl bromide (2.39 g; 0.022 mol) was added. The reaction mixture was worked up using methodology

identical to that described for the preparation of 60.

Recrystallization from benzene/hexane afforded 4.44 g (6 8 %)

of light yellow crystals mp 6 6 -6 8 °C. IR (KBr) 1765, 1710 cm ^; {^E-} NMR (CDClg)g 6.98-7.76 (m, BE, aromatic), 5.16

(s, 2H, benzylic), 3.7 (s, 3E, -CO2 CE3 ), 2.02 (q, 2E, CE2 ,

7.31 Hz), 0.87 (t, 3E, CE3 , J= 7.31 Ez). Calcd for the

AB part (benzylic) 6 ^ 3.66, 6 g 3.02 with J^g = 17.48 Hz; MS

(70 eV), m/e 326(M+>. 72

A n a l » Calcd for ^20^20^4* 73»60; H, 6.17. Founds

C, 73.70; H, 6.40.

5~Benzyloxy-2-ethyl-l-lndanone(62) . To a solution of

58 (3.26 g; 0.01 mol) in glacial HOAc (10 mL) was added 10

mL of dil HCl solution. The reaction mixture was worked up

using methodology identical to that described for the

preparation of Recrystallization from MeOH/H 20 afforded

1.86 g (70%) of colorless crystals mp 62-64°C. IR (KBr)

1700 cm ^ NMR (CDClg)g 6.96-7.75 (m, 8 H, aromatic),

5.15 (s, 2H, benzylic), 2.5-3.33 (m, 3H, ABC pattern of 2H-3

and H-2), 1.53- 1.97 (m, 2H, CHg)^ 0.99 (t, 3H, CH 3 , J= 7.31

Hz) .

Anal. Calcd for CigH^gO^: C, 81.16; H, 6.81. Found:

C, 81.30; H, 6.82.

Methyl 5-Benzyloxy-2-ethy1-1-oxo-2-indanpropionate(6 6 )

and Its Free Acid(44). To a solution of 5-benzyloxy-

2-ethyl-l-indanone ( 6_2 ; 1.33 g; 0.005 mol) in t-BuOH (25 mL) was added t-BuOK (0.22 g; 0.002 mol). The mixture was

stirred at room temperature under N£ for 5 h. Methyl

acrylate (0.9 g; 0.01 mol) was added and stirring continued

overnight. The reaction mixture was worked up using methodology identical to that described for 6 8 . Hydrolysis

of ester 6^ afforded a yellow semi-solid which was recrystallized from benzene/hexane yielding 1.1 g (65%) of 73

white crystals tup 155-157°C. IR (KBr) 3400, 1730, 1700

cm ^NMR (CDClg) 5 6.92-7.71 (m, 8H, aromatic), 5.12

(s, 2H, benzylic), 0.77-2.16 (m, 9H, aliphatic). Calcd for

the AB part (benzylic) 3.05, g g 2.83 with = 17.52 Hz;

MS (70 eV), m/e 338(M^^.

A n a l . calcd for ^ 2 1 ^ 2 2 ^ ^ ’ ^ » 74.53; Hj 6.55. Found:

C, 74.39; H, 6.59.

(trans)-5-Benzyloxy-2-ethy1-1-hydroxy-2-Indanproplonlc

Acid (4_2) ' To a solution of NaBH^ (0.114 g; 0.003 mol) In

0.2 N NaOH (1.0 mL) was added a solution of 5-benzyloxy-

2-ethyl-l-oxo-2-lndanproplonlc acid (4_4 ; 1.01 g; 0.003 mol)

In H 2 O (15 mL) containing NaOH (0.12 g; 0.003 mol). The

reaction mixture was worked up using methodology Identical

to that described for the preparation of 51.

Recrystalllzatlon from CHCI3 /benzene/hexane afforded 0.82 g

(80%) of white crystals mp 124—126°C. IR (KBr) 3400, 1690

cm“^; {^H-> NMR (CDCl^) g 6.78-7.72 (m, 8H, aromatic), 5.13

(s; 2H, benzylic), 4.75 (s, IH, C-1 H), 0.7-2.17 (m, 9H, aliphatic). Calcd for the AB part (benzylic) g ^ 2.95, 5 %

2.44 with = 15.26 Hz; MS (70 eV), m/e 340(M^).

A n a l . Calcd for C2 1 H 2 4 O 4 : C , 74.10; H, 7.10. Found:

C,74.52; E, 6.73. Chapter VII

PHARMACOLOGY

Introduction

Dibenzyloxyindanpropionic acid (j^) was found to inhibit

PGF2Q; “induced contractions of the isolated uterine strip with an IC50 of 3 . . This compound also exhibited in vivo protective effects against the abortifacient action of PGF 2 Q' in the mouse at an intramascular dose of 50 mg/

Kg^^*. The monobenzy loxyindanpropionic acid deivative A_6 tested previously showed slightly diminished activity against PGF 2q; “induced contractions of the uterus in vitro, with an IC50 of 7 xlO~^M^^^. The pharmacological activity of compounds 4^, and 51 was studied on the isolated mouse uterus stimulated with PGF 2 Q,.

Methods

Female albino CD-I mice, weighing 20-27 g, and in natural estrous, were sacrificed by cervical dislocation.

The uterine horns were isolated and prepared for isometric contraction recordings under 2 0 0 mg tension in oxygenated tissue baths maintained at 37°C. The composition of the bathing solution was (g/L): NaCl 8.046; KCl 0.2; CaCl2 *2 H 20

- 74 - 75

0.132; MgClg.SHgO 0.106; NaHCO^ 1.0; NaHgPO^.HgO 0.065;

dextrose 1.0. Following an equilibration period of 30 mins, -7 two 5-min control responses to PGF^^CIO M) were obtained,

followed each time by a 20-min washout period. The agent to

be tested was then added at a given concentration to the

bath and left in contact with the tissue for 5 mins prior to _7 addition of 10 M PGF^^. After recording the 5-min response

to PGF2 Q^in the presence of the test agent i the tissue was

washed for 20 mins with the physiological medium. Recovery

of the responsiveness of the tissue to ?GF 2a then -7 ascertained by adding 10 M PGFj^ for 5 mins. Finally, the

tissue was washed for 10 mins, the resting tension recorded

for 3 mins. Control uterine tissues were not exposed to the

test compound, but were otherwise treated similarly to the

test tissues. In these control tissues, the response to

PGF2 q; did not decline with repeated exposure. The integrat ed contractile force generated by PGF2 Q; in the presence of the test compound was expressed as a percentage of the mean of the two initial control responses to PGF 2 0 ; recorded prior to addition of the test agent. Recovery responses were expressed similarly.

The test compounds were dissolved in 100 pL 0.25 N

NaOH, and diluted with 100 pL distilled H 2 O to pH 7. PGF tromethamine and the test compounds were added in 10 pL volumes to the 10 mL tissue bath to obtain the desired concentrations. 76

Results and Discussion

Table 1 shows the effect of the test compounds on the

contractile response of the mouse uterus to PGF 2^ . None of

the test compounds had any effect at a concentration of

10 ^M. Compound 5J^ had no activity at a concentration of

10 and resulted in only a 2 0 % inhibition of the response

to PGF20; at the 1 0 ~^M concentration. Compounds 4^ and ^ at -5 a concentration of 10 M only weakly inhibited the PGFg^

response, and their approximate ICgg > 10 Recovery of

the tissue from the inhibitory effects of 45 and 5_^ was

essentially complete, while recovery from 44 was slightly

diminished.

Table 1. Effects of 4_5 and 5_1_ on PGF 2 Q-“induced

Contractions of the Mouse Uterus.

Compound n Cone. % Inhibition of % Recovery of PGF 2Q; response PGF2 q response

51 7 10“^M 0 91.70

8 lO'^M 20.39 103.70

45 9 1 0 "^M 12.16 92.20

11 10*4% 46.12 90.88

^ 1 lO'^M 27.29 69.32

6 1 0 "^M 44.42 70.39 77

The results indicate that the monobenzyloxyindan- propionic acids are weaker than dibenzyloxyindanpropionic acid in their ability to antagonize PGF^g induced uterine contractions.

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