HYPOCHOLESTEROLEMIC AGMOB: COMPOUEDS EBIATED

TO ETHYL a-( 4-CHLOEOPHEHOXY) -a-METHYLPROPIONATE

DISSERTA.TIOE

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

t By

Robert E . Hackney, B.S., Pharmacy

******

The Ohio State University 1970

Approved by

Adviser Department of Pharmacy ACKNOWIÆDGMENÏB

I vi'sh to express my sincere appreciation to my adviser,

Professor Donald T. Witiak, without whose guidance and encouragement this would not have heen possible.

I am. also deeply grateful to Dr. Robert Ober for his very valuable suggestions which added much to this work.

I also want to thank my fellow graduate students whose friendship and discussion have been most helpful.

To my parents, without whose love, understanding, and con­ tinual encouragement this tenure would not have been possible, saying thanks is completely inadequate.

ii v m

October l6, 1 9 ^ 2 ...... B o m - Wellington, Kansas

1 9 6 5...... B.S. Pharmacy, The University of Kansas, Lawrence, Kansas

1 9 6 5 - 1 9 6...... 7 Research Assistant, The University of Iowa, Iowa City, Iowa

1 9 6 7 - 1 9 7...... 0 Research Assistant, The Ohio State University, Colxmbus, Ohio

PUBLICATIONS

Hypocholestérolémie Agents. Compounds Related to Ethyl a-(4-Chloro- phenoxy ) -a-Methylpropionat e.

D. T. Witiak, T. C. -L. Ho, W. E. Connor and R. E. Hackney, J. Med. Chem., n, IO8 6 (1 9 6 8).

Inhibition of Cholesterolgenesis In Vitro by Chlorophenoxyacetic Acids. Effect of a-Methyl Groups.

D. T. Witiak, R. E. Hackney and M. W. Whitehouse, J. Med. Chem., 2^, 6 9 7 (1 9 6 9).

FIELDS OF STUDY

Major Field; Medicinal Chemistry

iii TABLE OF CONTENTS

P age

ACKNOWLEDGMENT...... ii

VITA ...... iii

LIST OP T A B L E S ...... v

LIST OF FIGURES ...... vi

Chapter

I. INTRODUCTION ...... 1

II. RESULTS AND DISCUSSION...... 12

III. Chemistry Biological

III. EXPERIMENTAL ...... 27

Chemical Biological

IV. SUMMARY ...... 58 V. REFERENCES...... 60

IV LIST OP TABLES

Table ' Page

1. The Activity ^ Vivo of a Series of Ethyl a- ( 4-Substitnted-Phenoxy ) -a- Methylpropionate Analogs...... 26

2. Effect of Compounds 2, D-4 and L-4, and 37 Added In Vitro on the Incorporation of Acetate- 1-l^C Into Nonsaponifiable Material of Rat Liver Homogenate...... 31

3 . Effect of 2; D-4 and L-4 Added ^ Vitro on the Incorporation of”Mevalonate-2-l^C Into Nonsaponifiable Material in Rat Liver Homogenate...... 31

4. Effect of 6c, Jc, and l4 Added ^ Vitro on the Incorporation of”Mevalonate-2- l^C Into Nonsaponifiable Material in Rat Liver Homogenate...... 34

5« Ethyl a- ( 4-Subst ituted-Phenoxy ) -a-Methylpropionate s . . . 37 LIST OF FIGURES

Figure Page

1. The Optical Rotatory Dispersion and Circular Dichroism Curves for Ethyl L-(- )-a- ( 4-Chloro- phenoxy) Propionat e...... 15

2. The Configurational Relationships of L-Thyxoxine, a-(4-Chlorophenoxy)-a-Methylpropionic Acid, L-f-)-a-(4-Chlorophenoxy)propionic Acid and D-(+)-a-(U-Chlorophenoxy)Propionic A c i d ...... 28

3 . Dose-Response Curves for thé Per Cent Inhibition of Incorporation of Mevalonate-2-^^C Into Nonsaponifiable Material in Fortified Rat Liver Homogenate by Acids 2, L-(-)-^, U-(+)-4 and ^ • • • • • 32

14-. Dose-Response Curves for the Per Cent Inhibition of Incorporation of Mevalonate-2-l^C Into Nonsaponifiable Material in Fortified Rat Liver Homogenate by Acids Sç, Tç,and ...... 35

VI CHAPTER I. IKTR0DUCÎEEO1Î

Atherosclerosis is the most common form of arterial disease today. It is a complex metabolic disorder marked by the development of lesions in large or medium-sized arteries and it is characterized by the accumulation of lipid material in the intimai and inner medial layers of 1 2 the arterial wall. ’ Several general principles concerning the disease O are evident: (l) Age dependence is not obligatory. (2) Incidence of the disease varies with population and geographic area. (3) A direct correlation between development of the disease and elevated blood lipid levels exists. (4) Arterial wall tension and other arterial injuries are important. (5) Females are less susceptible to the disease.

The exact mechanism of the disease remains unknown despite extensive investigation in the area. The factor most readily correlated k to the disease is elevated cholesterol levels. Cholesterol is present in the free and esterified form in the atherosclerotic plaque; phospho­ lipids, cerebrosides, a small amount of neutral fat, and dihydrochol­ esterol are also present Although normal tissue and plasma contain small amounts of dihydrocholesterol, atherosclerotic lesions contain approximately 10 per cent of this substance.^ Various animals fed a high-cholesterol diet develop experimental lesions closely resembling 7 those seen in man. They are similar in both morphology and location.

The normal artery presents a barrier to the influx of serum cholesterol; gradual breakdown of this barrier in the presence of hypercholesterolemia

1 allows for the accumulation of lipids and the formation of atheromatous g lesions. Metabolic defects in the synthesis, transport and utilization of cholesterol may therefore he important factors in the pathogenesis of 9 the disease.

According to Guhner and lingerieider^^ regulation of hlood cholesterol appears to offer the most promising approach to the preven­ tion of atherosclerosis. It is possible to cause marked reductions in plasma lipoprotein concentrations by dietary measures. However, diets are inconvenient and many patients have difficulty adhering to them.

Pharmacological agents which lower blood cholesterol concentrations in patients and do not exhibit harmful side-effects should be very useful.

Investigators have found that a variety of compounds inhibit the biosynthesis of cholesterol^^ or influence cholesterol metabolism in some other way. 12 For example, various derivatives of mevalonic acid and biphenylvaleric acid inhibit the incorporation of acetate and/or mevalonate into cholesterol. N-Ethylmaleimide, p-chloromercuribenzoate,

Triparanol^^, and AY-99^^^^ inhibit the biosynthesis of cholesterol from squalene at various stages in the biosynthetic pathway. Thyroid hormones cause increased conversion of cholesterol into bile acids. Estrogens decrease the concentration of cholesterol attached to the ^-lipoprotein fraction.

We are interested in the compound ethyl a-(4-chlorophenoxy)- a-methylpropionate (la)In I9 6 2 Thorp^^ reported that a combination of la and androsterone reduce serum lipid levels in experimental animals. I Comparative studies on the mixture and la alone show both to be equally effective in lowering elevated serum cholesterol levels and probably also in reducing elevated triglycerides. 17 . Following these reports the hypocholesterolemic and hypolipidemic effects of la have heen the sub­ jects of numerous publient ions, but the mechanism of action of the compound still remains obscure. Two theories receiving the most atten­ tion have been the thyroxine release mechanism^^'and inhibition of 21 cholesterol biosynthesis.

C(CH3 )2 C0 2 R

la, R = C 2H^

2, R = H

Let us first consider the significance of thyroxine in metab­ olism. Hypothyroidism in humans results in elevated concentrations of plasma cholesterol; treatment of such patients with thyroid hormone lowers plasma cholesterol concentration to normal. 22 '21 I n myxedematous or hyperlipedmic patients thyroid hormones induced a fall in serum chol- esterol levels. 24,25 ' The mechanism by which thyroid hormones lower blood serum cholesterol levels appears to involve both the biosynthesis and degradation of cholesterol. Thyroid hormones lower serum cholesterol in vivo by increasing cholesterol catabolism and excretion. As shown by

Rosenman and co-workers 2 6 , there is a decreased output of biliary cho­ lesterol in the hypothyroid rat. Conversely, there is an increase in 27 biliary cholesterol in the hyperthyroid rat. Erickson demonstrated lowering of the total bile acid excretion in the hypothyroid rat. In vitro studies were used to show thyroxine inhibits the biosynthesis of l4 29 cholesterol from C-acetate in rat liver homogenates. 2 8 ' Thyroxine h and thyroxine analogs also lower serum and liver cholesterol concentra­ tions in euthyroid; cholesterol-fed rats.^^-’^^

It has become increasingly evident that the interaction between thyroid hormones and serum proteins has an important influence on their 32 distribution, degradation, excretion and action. A body of evidence is available suggesting both the hypocholesterolemic effect and the rate of degradation of thyroxine are functions of the concentration of un­ bound hormone.The concentration of free thyroxine in human blood is extremely low; more than 9 9 * 8per cent of total thyroxine in blood is bound by proteins. Any compound which depresses protein binding of thy­ roxine, either by changing the affinity constant or by reducing the con­ centration of available unoccupied binding sites, may potentiate the action of endogenous thyroxine and alter its rate of metabolism. Accord­ ing to the thyroxine release mechanism, 3^ may undergo rapid to vivo hydrolysis to the free acid^^ 2 which exerts its effect, at least in part, by displacement of thyroxine from its binding proteins in the plasma followed by redistribution of the hormone between the plasma and liver.The result is presumed to be increased lipid metabolism^^ with concomitant lowering of serum lipid levels because of the hyperthyroid effect in the liver.Although studies with euthyroid patients reveal that la produces no significant change in the glycine to taurine con­ jugate ratio with biliary bile acids as is the case with hypothyroid patients treated with thyroid hormones 34 , the authors 35 suggest these observations are not conclusive negative evidence for the thyroxine release mechanism. Preliminary experiments indicated la resembles 2,4- dichlorophenoxyacetie acid which alters the physiological distribution 5 of thyroxine hut leaves the concentration of free thyroxine in the plasma

and output of pituitary thyrotropin essentially unchanged, producing

these effects principally hy an action on the liver. 37 Recently, Westerfield and co-workers reported the effect of

la on rat liver a-glycerophosphate dehydrogenase (g PD) and malic enzyme.

The size and number of mitochondria as well as several enzymes associated with liver mitochondria,. GPD and malic enzyme, are increased in rat liver Q O hy 2^. Previous studies have demonstrated a high degree of specificity

of thyroxine and its analogs for GPD and malic enzyme. This suggests a

thyroidal effect hy ]a. 39 Feeding of la to thyroidectomized rats in­

creases the liver GPD insignificantly. Injection of 1.0 |ag of thyroxine

to intact or thyroidectomized rats fed the hasal diet in which their

liver GPD=25-^5 units results in an increase in liver GPD slightly ahove the euthyroid level in hoth animals. However, injection of 1.0 |j.g of thyroxine to intact or thyroidectomized rats fed the hasal diet in which their liver GPD=25-^5 units gave values of 80-125 units in the presence of la in hoth types of rats. These results are summarized as follows:

(1) Compound la exhibits little or no effect on liver GPD in the absence

of thyroid hormone; therefore compound la is not thyromimetic per se.

(2 ) The thyroid gland is not involved in the enhancement of exogenous thyroxine activity hy la. Conversion of a small thyromimetic stimulus

into a large GPD and malic enzyme response was confined largely to the

liver. This suggests two possibilities: (l) Circulating thyroxine is 1].0 concentrated in the liver hy displacement from the plasma proteins.

(2 ) The drug inhibits the normal destruction or removal of thyroxine hy the liver. However, the following points discount hoth of these 6

theories. First, the total amount of thyroxine in the plasma of a 100 g

rat is less than 0 . 5 Mg^ while the effect of the drug is equivalent to a

dose of 4.0-8.0 pg of thyroxine per day. Second, any thyroxine immedi­

ately displaced from the plasma proteins by the drug would not sustain

an elevated GPD for 6 weeks since the thyroxine would be metabolized

before this time. Further, if were simply blocking the destruction

of thyroxine, injection of small doses of the thyroid hormone into rats

receiving la should give an increased GPD response. This was not

observed.

Another possible mechanism of action of 3^ involves stimula­ tion of protein synthesis.The total liver GPD was increased as

rapidly as 3^ alone as the stimulus produced by 40.0 (ug thyroxine, and this increased GPD can be attributed to new protein synthesis. Hence, the small stimulus for new protein synthesis normally provided by endog­ enous thyroid hormone became a large stimulus for new protein synthesis in the presence of la. Some thyroid hormone was required to provide the initial stimulus, but once initiated, la had the effect of keeping the synthetic machinery "open"; i.e. it prevented the normal cutoff in the synthetic process. The maximal rate at which large doses of thyroxine normally increased the liver GPD was limited by the normal cutoff mech­ anisms, but when the latter were blocked by 3a, this rate was increased still further. Inhibition of the GPD response to ^ by protein synthesis inhibitors such as actinomycin, cycloheximide and ethionine supports the l|.l stimulation of protein synthesis theory.

The relationship of changes in liver GPD or malic enzyme to lowering of blood lipids by la remains to be determined. An increase 7 in liver GPD activity should decrease the amount of glycerophosphate substrate available for lipid synthesis which in turn could limit the rate at which triglycerides are synthesized and released from the liver h2. into plasma. Malic enzyme catalyzes formation of L-malate, which could then he oxidized to acetyl CoA and other metabolites leading to increased 1|.Q lipid levels. Regardless of mechanism, la has a thyroxine-like effect which alters lipid metabolism.

Inhibition of cholesterol biosynthesis is another possible mechanism by which 3a could lower blood serum cholesterol. Cholesterol is or may be a precursor in the biosynthesis of various IT-keto- or 1 7- kh ketogenic steroids, such as androsterone, cortisol and cortisone.

Inhibition of cholesterol biosynthesis by 3a should result in a decrease in urinary 1 7-keto- or 1 7-ketogenic steroids; however, studies in humans demonstrate that ^ produces no significant decrease in these steroids.

It has been suggested that since most of the patients in these studies were hypercholesterolemic, steroid production is not dependent upon a 46 reduction of the serum cholesterol from elevated to normal levels.

However, when given to rats at 0.2 per cent of the diet, la significantly decreases the secretion of adrenal steroids and vitro inhibition of steroidogenesis is dose related to the reduction of adrenal steroids and the hypocholesterolemic effect However, the specific site of in­ hibition of cholesterol biosynthesis has not been determined. Thorp and

Waring^^^ report 3a may inhibit acetate incorporation into cholesterol by rat liver slices in vitro. Gould and co-workers 48 conclude the site of ^ inhibition is between acetate and mevalonate. Other reports sug- 21d 49 gest la blocks cholesterol biosynthesis after mevalonate. 8

In the ethyl a-phenoxy-a-methylpropionate series, the mech­ anism of action has heen investigated only with la, which is reported to he the most active analog.If, however, analogs were to he found having activity ^ vivo equal to subsequent parallel vitro studies may serve to differentiate between possible mechanisms of action on a structural basis.

This thesis is concerned with the synthesis and biological evaluation of a series of ethyl a-(li-substituted-phenoxy)-a-methylpro- pionates (l) and related analogs and esters having various electron- donating and electron-withdrawing substituents at the 4-position. Sub­ stituents are varied in the 4-position to determine their effect on hypocholesterolemic activity. Other analogs are prepared for purposes of identifying stereochemical relationships to drug activity. Analogs of interest for studying electronic effects are identified by struc­ ture 1 where R = C1, CH^, CH^O, H and CN.

R— 0—c(CHg)2 C0 gC2 H^

1

R=C1 W, R=H

R = C H 3 R = C N

Ic, R =01130

Compounds of interest for studying stereochemical aspects of drug action are identified as follows. Removal of one of the gem- dimethyl groups of la affords the desraethyl analog 3* Resolution of

3 affords the D and L enantioraorphs. The two stereoisomers are desired for biological evaluation to deteimine minimum structural requirements

for both iu vivo and i^ vitro. The effect of these compounds on the in­

corporation of acetate-l-^^C and mevalonate-2-^^C into nonsaponifiable

material, squalene and cholesterol is discussed. Certain albumin bind­

ing studies involving these two (optically) isomeric acids 4, the gem-

dimethyl compound 2 and related analogs are presented. These studies

are discussed relative to the thyroxine release mechanism which is pre­

sumed to involve displacement of endogenous L-thyroxine (5) from its

preferential binding site on rat albumin.

Cl— 0— CH(CH^)COgR

3 , R=C2 H^

4, R=H

« 2— C— COgH

The synthesis of other analogs of la is also discussed. These

compounds are of interest for studying stereochemical requirements for

biological activity. Certain benzodioxanes and benzofurans of the struc­

ture types 6 and J, respectively, represent the tying up of one of the methyl groups of the gem-dimethyl compound to the ortho position of the aromatic ring. Successful synthesis of these compounds which may con­ tain either a methyl group or a hydrogen atom on the a-carbon (as desig­ nated by E) may also be resolved into their respective enantiomorphs and 10

studied biologically. As is the case with compound la, ethyl esters of

these compounds are of interest for studies ^ vivo. The free carboxy

compound, like the situation for la, may be the active metabolic product

of the ester in vivo, and will be used for studies in vitro.

R COgR'

O O'

6a, R=H, R '=CgH^

6b, R=CH^, R

6c, R = H , R '=H

6d, R=CH^, R'=H

COgR'

Ja, E==H, R'==C2 H^

7b, R=CH^, R '=02^^

7c, R=H, R'=H

Jà, R=CH^, R'=H

In this thesis we also discuss some of our studies directed toward the synthesis of respective homologs 8, 9> and of compound la, the D- and L-desmethyl analogs 3> and the benzofuran system 7 having a methylene group inserted between the asymmetric carbon atom and the

carboxyl group. These compounds are of interest because of their apparent relationship to L-mevalonic acid (U) and because it is well 11 known that the gem-dimethyl compound la^ as well as the D- and L- desmethyl isomers 3 , inhibit cholesterol biosynthesis between mevalonate and squalene. It will become apparent from the discussion of our exper­ imental results why homologs of structure la are of interest. CH3 I ^^CHqCOq R

CH3

8a, R=CgH^

R = H

H I ^,-CHgCOgR

/\

9a, R==C2 H^

9b, R = H

Cl

10a, R=H, R ’=C 2H^

10b, R=i=CH3 , R ' = H

CHpCHpOH I y^^^'CHgCOgH HO

11 CHAPTER II. RESULTS AHD DISCUSSION

Chemistry

Ethyl a-(4-chlorophenoxy)-a-methylpropionate (l^) is prepared hy condensation of 4-chlorophenol (l2 ) with acetone and chloroform in the presence of sodium hydroxide; subsequent estérification of the re­ sulting acid (2 ) affords (la). Other ethyl a-(4-suhstituted-phenoxy)- a-methylpropionates (l) are similarly prepared in 3 0 -3 5 per cent over­ all yield. 51

0

Cl— CH^-C-CHg + CHClj

12

(1)NaOH (2 )HC1 /H20

Cl— — 0-C(CH3)2C02H

HgSOi^/CgH^OH

Cl— ^ O y - O - C C C H g )2C02CgH^

la

Ethyl a-( 4-chlorophenoxy)propionate (^) is readily synthe­ sized hy condensation of 4-chlorophenol (1 2 ) with ethyl a-hromopropionate

12 13 Cp (1 3 ) in the presence of potassium carbonate. Hydrolysis of 3 yields

the free acid 4. Resolution of 4 with (-)-brucine affords the (-)acidj^^

resolution with (+)-yohimbine yields the (+)enantiomorph.^^

Cl— ^ Q ^ -—OH + CHgCH(Br)C02C^^

12 13 KgCOg

Cl-~^(^ y — 0-CH(CHg )C02C^^

(1)NaOH (2 )HC1 /H20

-CH(CHg)C0 2 H

(-)-brucine (+)-yohimbine

(-)& (+)4

Analysis of the optical rotatory dispersion (oKD) and circular dichroism (CD) curves for both the (+) and (-) acids, liberated after three to five recrystallizations of their respective salts to constant rotation at the sodium D line, confirm the optical purity of the com­

pounds and reveal their absolute configurations to be D-(r ) and L-(s),

respectively. 8 2 The ethyl esters prepared from the optically pure acids Ill- gave OKD and CD curves similar to those observed for the respective acids. OKD and CD spectra examples for aster L-(-)-3 are illustrated in Figure 1. Esters and acids of the D- (+ ) - configurât ion exhibit a positive Cotton effect which is the mirror image of those shown in

Figure 1. For these compounds the optically active chromophores result in peak formation at 288-290 mp. (positive shoulder at 283-284 mp), a trough at 264-205 ny, and a positive plain dispersion curve between 2Ô0 82 and 235 mp.. These data are in agreement with the work of Sjoberg who observed a positive plain dispersion curve in the region between TOO and

300 for a series of D-a-phenoxypropionic acids and amides. Instru­ mentation limitations did not allow Sjoberg to observe the remaining spectrum at shorter wavelengths than 3 OO mp.

Ct-(4-Chlorophenoxy)-a-raethylbutyric acid (]A) is prepared by condensation of 4-chlorophenol (12) with 2-butanone and chloroform in the presence of sodium hydroxide; acidifcation affords in 25 per cent 55 yield. Attempts to resolve were unsuccessful.

0 II iH + CH3 -C-C2 H5 + CHCI3

12 (1)NaOH (2 )HC1 /H20

Cl— ^ Q ^ — 0 -(CH3 )(C2 H5)C0 2 H

14 10

CRD

-10 o> -e-

-15

-20

-25

200 400

Figure 1 The Optical Rotatory Dispersion and Circular Dichroism Curves for Ethyl L-(-)-a-(U-Chlorophenoxy)Propionate j-j VI l6

l,4-Benzodioxan-2-car'boxylic acid (6c) is prepared for evalua­ tion of hypocholesterolemic activity. Catechol (l5) and ethyl 2,3- dihromopropionate (l6) are refluxed in acetene in the presence of potas­ sium carbonate affording ethyl 1 ,4-benzodioxan-2-carboxylate {§a)i this 56 compound is subsequently hydrolyzed to 6c in 6j per cent overall yield. ©c-

!02 H

6c

5-Chloro-2,3-di^y<^i’o-2-benzofurancarboxylic acid (Tç) is pre­ pared as follows: condensation of 5-chlorosalicylaldéhyde (1 T) and diethyl bromomalonate (^) in the presence of potassium carbonate affords ethyl 5-chloro-2-benzofurancarboxylate (3^) in Jd per cent yield. Hy- 57 drolysis affords the free acid (20) in 57 per cent yield. Reduction of 2 0 with sodium amalgam affords the dihydro derivative Jc in 7 5 per cent yield.

BrCHCCOgCgHc)^ JsX” '

17 18

K2 CO3 17 oTY

(1)laOH (2 )HC1 /H20

COgH

(l)lfa(Hg) (gjHCl/HgO

CdgH

Ethyl 5-chloro-2,3“dihydro-2-methyl-2-'benzofurancarboxylate

(7b) is also of interest since this compound is a a-methyl to ortho aromatic connected analog of 3^. The first synthetic approach which we attempted involved méthylation of ethyl 5-chloro-2;3“b,ihydro-2- henzofurancarhoxylate (Ta) • 5-Chloro-2,3-dihydro-2-'benzofurancar'boxylic acid (Tç) is converted to the ethyl ester (Ta). However, attempts to methylate 7h using methyl iodide were unsuccessful. The use of sodium hydride in dimethylfomamide or dimethyl sulfoxide,, or sodium ethoxide in ethanol affords starting material.

Cl Tc 18

Cl- Ta

Cl Tb

A second synthetic scheme involved Dieckman condensation of ethyl a-(^-chloro-2-carhoethoxyphenoxy)propionate (2U). This would afford 5-chloro-2-methyl-2-carboethoxy-3(2H)-henzofuranone (25) which, m i ^ t subsequently be reduced to the desired product Tb.

C^^OH/HgSOlj.

COgCgH^

CH^CH(Br)C02C2H^ 2| KgCO^

O-CHfCHjïCOgCgH^

C 1 'foT' ^ \ ^ C 0 C H. 2h ) base 19 CH

Cl 25 [H] CH3

Cl 7b

5-Chlorosalicylic acid (21) served as starting material and is converted to the ethyl ester^^ 22 which is condensed with ethyl a-broraopropionate

(2 3 ) in the presence of potassium carbonate. This affords ethyl a-(4- chlüx’o-2-carbuethoxyphfeuûAy)prüpiouatc (24) in 93 cent yield. At— . tempts to carry out this condensation using sodium hydride or sodium ethoxide as bases yielded phenolic compounds. Evidently, elimination of the ethyl propionate side-chain occurs. during the reaction and this approach was therefore abandoned.

Another synthetic scheme involved cyclization of diethyl 2-(4- chlorophenoxy)-2-methylmalonate (2%) to 5-chloro-2-methyl-2-carboethoxy-

3(2H)-benzofuranone (29)»

■— OH + CH^C(Br)(C02C2H^)g

12

KgCOg

C(CH3)(C02C2H^)2 20

U-Chlorophenol (12) is condensed with diethyl 2-‘bromo-2-inethylraalonate

(2 6) in the presence of potassium carbonate affording diethyl 2-(4- chlorophenoxy)-2 -methylmalonate (2 %) in 55 per cent, yield. However, attempted cyclization of ^ according to a method of Fieser and Fieser 59 using a sodium chloride-aluminum chloride melt affords a black tar. No small molecules are isdatable from this material. Attempted hydrolysis of ^ results in decarboxylation to 2-(4-chlorophenoxy)propionic acid (4).

5-Chloro-2-methyl-2-carboethoxy-3(2H)-benzofuranone (^) is prepared by méthylation of 5-chloro-2-carbosthoxy-3(2H)-benzofuranone

(30 )• Ethyl 5-chlorosalicylate (22) is condensed with ethyl 2-bromo- acetate (2 8) in the presence of potassium carbonate; this affords ethyl

4-chloro-2-carhoethoxyphenoxyacetate (2§) which is eyelized to 5-chloro-

2-carboethoxy-3(2H)-benzofuranone (3 0 ) in 72 per cent overall yield.

Various attempts to C-methylate compound ^ employing methyl iodide and sodium ethoxide yielded starting material. Use of methyl iodide and sodium hydride in dimethyl sulfoxide, or dimethyl sulfate and sodium ethoxide in absolute ethanol affords the 0-raethylated product ethyl

5-chloro-3-methoxy-2-benzofurancarboxylate ( ^ ) . C-Méthylation is accomplished in 50 per cent yield by heating 5-chloro-2 -carboethoxy-

3 (2H )-benzofuranone (3 0 ), methyl iodide, sodium ethoxide and absolute ethanol in a sealed Pyrex tube at 100° for 2 hours. The isolated compound is characterized by means of nmr spectroscopy; the C-methyl resonance signal appears as a sharp singlet at l.?4 6, whereas the 0 - methyl resonance signal for the 0 -methyl compound ^ prepared previously appears as a sharp singlet at 4.23 S. 21

+ BrCHgCOgC^H^ C 1Igl" ' \ x ^C02C2H5 22 28

K2CO3

OCH2CO2C2H5

C 2H3

COgCgH^

NaOCgH^/CHgl • NaH/CHol/DMSO IQO/ssalcd tube OT* (CH3)2SOl|./lIaOC2H5

"0s.,_C02C2H$

Cl 25

Several approaches involving the reduction of 5-chloro-2- methyl-2 -carboethoxy-3 (2H )-henzofuranbne (25) to ethyl 5-chloro-2,3- dihydro-2 -methyl-2 -'benzofurancarbo:qrlate (7b) were studied. The first approach involved formation of the ethylene dithioketal of 25 followed by Raney nickel desulfurization. Apparently, enol foimation is necessary for ethylene dithioketal formationthis is a likely explanation for the lack of success with this reaction since for compound ^ no enol is possible. 22

Another approach involved reduction of the p-toluenesulfonyl-

hydrazone of ^ using sodium horohydride. ■ Formation of ethyl 5-chloro-

3-(p-toluenesulfonylhydrazone)-2-methyl-2 “'benzofurancarboxylate (^) is

accomplished by condensation of 5-chloro-2-metl%rl-2 -carboethoxy-3 (2 H)-

benzofuranone (2 ^) with p-toluenesulfonylhydrazide (3 1 ) in the presence

of hydrochloric acid catalysis. Reduction of ^ with sodium borohydride

was not successful; the products of the reaction have not been charac­

terized.

CHo

CH3 — SO2NHNH2 Cl 31

HC1 /C2H 5OH

CO2C2IÎ5

:B-EH-02S

A third approach involves reduction of 5-chloro-2 -methyl-2-

carboethoxy-3(2H)-benzofuranone (25) under Clemmenson conditions. Com­

pound ^ is dissolved in acetic acid and stirred overnight with hydro­

chloric acid and water, followed by warming on a steam bath for 1 hour.

Again, the desired compound was not isolated. A discussion of the re­

sults of this reaction and products formed will be the subject of another

communication. 23

Another compound desired for testing for hypocholesterolemic

activity is 5-chloro-2,3-^ihydro-2-'benzofuranacetic acid (lOc). One

synthetic approach could involve reduction of 5-chloro-2 ; 3-&ihydro-2 -

■benzofurancarhoxylic acid (^) to 5-chloro-2 ,3-dihydro-2 -hydroxymethyl-

benzofuran (33)* Subsequent formation of the methane sulfonate deriva­

tive of followed by the displacement of the methane sulfonate group

by cyanide ion, and hydrolysis would afford the free acid 10 c.

CHoOH COoH LUIHI^ Cl Cl 7c 33

CH3SO2CI pyridine

(^^CHgO^SCH^ (l)KCN Cl *C2)HC1/H20 10 c

5-Chloro-2,3-dihydro-2-henzofurancarboxylic acid (Tc) is reduced with

lithium aluminum hydride affording 33 in TO per cent yield. Alcohol ^

is converted to 5- chloro- 2 ,3 -dihydro- 2 -hydr oxymethylbenz ofuran methane-

sulfonate (_3^) by stirring with methanesulfonyl chloride in pyridine.

Displacement of the methanesulfonate group with cyanide ion was attempted

according to a method of Pelizzou and Jommi 62 employing potassium cya­ nide, potassium iodide, dimethyl formamide and water. A black oil re­

sults which shows no triple bond absorption in the infrared. 2 h

Another synthetic approach involves foimation of the grignard reagent of 5 - chloro- 2,3 - dihydro - 2- chloromet hylbenz ofnran (3^). Subse­ quent treatment with carbon dioxide followed by acidification should afford 5-chloro-2j3-dihydro-2-benzofuranacetic acid ( 10c). In fact, ^ is obtained by reaction of 33 with thionyl chloride in pyridine. How­ ever, all attempts to obtain the grignard reagent results in isolation of starting chloride 33-

X^CHgOH

SOCI2 pyridine

Cl

A third reaction sequence involves formation of 5-chloro-2,3- dihydro-2-benzofuranacetic acid (lOc) from 5-chloro-2,3-dihydro-2-benzo- furanoyl chloride (36) using the Arndt-Eistert synthesis. Acid 7c is converted to the acid chloride 36 by re fluxing with thionyl chloride in benzene. Treatment of the acid chloride ^ with freshly distilled dia- zomethane yields an oil which is treated with silver oxide in absolute ethanol. The distilled product is not the desired compound and remains to be characterized. 25

Cl Tc SOCI2 benzene

\ ^ C 0C1

A modification of the Arndt-Eistert synthesis, proposed by

Newman and co-workers, ^ was next investigated. The oil obtained from treatment of the acid chloride with freshly distilled diazomethane is dissolved in absolute methanol and treated with silver benzoate in tri- ethylamine. A rapid evolution of gas ensues and the reaction mixture turns dark black. Characterization of the product of this reaction will be discussed in a future communication.

Biological

In the series of ethyl a- (4-substituted-phenoxy)-a-methylpro- pionate analogs of la, only the unsubstituted compound ^ exhibits sig­ nificant hypocholesterolemic activity (p<0 .0 5) in normocholesterolemic rats.^^ The activity of Id compares reasonably well with that of la when the drug is administered orally mixed with the diet (Table l). No significant hypocholesterolemic activity is observed at O.I5, 0.20, and

0 .5 0 per cent drug in the diet for the 4-CHg (lb), CH^O ( ^ ) , and CN

(le) analogs. 26

Table 1 The Activity ^ Vivo of a Series of Ethyl a-(^- Substituted-Phenoxy)-a-Methylpropionate Analogs

Wt, g. Serum ^ Drug After Cholesterol, Compound in Diet wt, g^ 12 Days^ mg^c

Control I — — 536 522 104 ± 5^

la 0 .1 5 450 471 74 + 4 0.20 514 471 64 + 4 0 .5 0 489 455 71 ± 5

Id 0 .1 5 415 432 84 ± 6 0.20 460 481 81 ± 7 0 .5 0 46o 48l 60 ± 4

Control II — — 585 591 104 ± 7

DL-3 0.20 0.20 600 607 87 ± 9 0 .5 0 6l4 591 85 ± 6

0.20 583 523 98 + 8 0 .5 0 588 511 83 ± 7

0.20 627 595 79 ± 7 0 .5 0 639 608 72 + 8

Ethyl L-(- )-a-(4-chlorophenoxy)propionate (^) exhibits signif­

icant hypocholesterolemic activity (p<0.05) in normocholesterolemic rats.

While the apparent hypocholesterolemic activity of the D and DL compounds

is not significant, the activity of the L isomer compares favorably with

the hypocholesterolemic effect of ^ at similar dosages.

The general toxicity of these compounds is observed by measur­

ing a gain or loss of weight. All rats are weighed before and after the

12-day feeding period. The entire diet is consumed by the rats. No rela­ tionship exists between the loss or gain of weight, toxicity and the hy­

pocholesterolemic effect. Control groups vary by about 10 g. While the 27

average weight loss for rats on a diet containing 0.20 and O .50 per cent

L-(-)-3 or la is ahout the same (30-35 g), rats on the D-(+)-3 diet lost

an average of 20g indicating this compound may he less toxic than la.

These studies suggested to us that the minimum structural

requirements for analogs of compound 1^ having activity are (a) no func­

tional group in the para position of the phenyl ring, and (h) only one methyl group on the a-carhon hut in the correct configuration L-(-)-3*

It may he possible to obtain a greater discrimination of activities with

hypercholesterolemic rats or other animals. Using normocholesterolemic

rats the compounds tested could not he classified as significantly active or inactive at the three dosage levels employed.

If one assumes that an aryl ether oxygen may interact with a protein in a manner not unlike an amino group, then L-(-)-a-(U-chloro- phenoxy)propionic acid (4), the possible metabolic product of L-3, is biologically most closely related by configuration to L-thyroxine (5).

If one further assumes that L-thyroxine is bound to protein by means of its carboxyl, amino and aryl groups, one explanation for the greater activity of L-3 might be that the L acid (4), formed after hydrolysis in vivo, more readily competes with L-thyroxine for an asymmetric bind­ ing site on a protein by means of the carboxyl, ether oxygen, and methyl groups. In the case of the D acid (4) the methyl group apparently has the wrong configuration; with the gem-dimethyl acid 2, one of the methyl groups must have the right configuration. The configurational relation­ ships of the various compounds are shown in Figure 2. 28

H COgH

L-thyroxine

A7y={ 3, 5-diioàe-U-hydroxy- phenoxy)-3,5-diiodophenyl

f s / % -\ A----CO 2H C l —

2, a- ( 4- chlorophenoxy ) -a- methylpropionic acid

y---V X. ---- COgH Cl—

4, L-(-)-a-(4-chlorophenoxy)- propionic acid

CH ^ COgH Cl-

4, D- (4- )-a- ( 4-chlorophenoxy ) - propionic acid

Figure 2 The Configurational Relationships of L-Thyroxide, a- (4-Chlorophenoxy)-a-Methylpropionic Acid, L-(-)- a-(4-Chlorophenoxy)Propionic Acid and D-(+)-a-(4- Chlorophenoxy)Propionic Acid 29 However; there are several alternative possibilities why the D

and L esters show differing hypocholesterolemic activity: (l) the D acid

is formed less readily from the ester to vivo, and/or (s) it is bound to

a greater extent than the L or gem-dimethyl compound to protein sites of

losS; and/or (3 ) the D acid is excreted more readily, and/or (^) the L

acid acts mainly as a stereoselective Inhibitor of cholesterol biosyn­

thesis not related to L-thyroxine,

In vitro studies were next carried out to determine which of

these proposals or combinations of proposals represents a most likely

explanation for the differences observed for drug activity in vivo.

Experiments in this laboratory have shown that like la, which readily

undergoes in vivo and in vitro hydrolysis to 2, esters L-3 and D-3 are

readily hydrolyzed to their respective acids L-4 and D-k by rat liver,

serum and intestinal preparations. 65 Consequently, differences in the

activity in vivo of the two esters cannot be explained merely by a dif­

ference in apparent half-life.

Albumin binding studies utilizing la and the two optically

isomeric acids 4 have been carried out by Witiak and co-workers.

The acid D-h binds to a greater extent than acid L-k to rat albumin and

to the same extent as 2. The observed disparity to the binding of D-4

and L-4 does not support the hypothesis that clofibrate acts to lower

cholesterol levels by displacing thyroxine from its binding site(s) on

rat albumin. The free acid L-4 is the active isomer to vivo in rats; 6i|. the D-isomer is not significantly effective, but it is the D-acld 36 which binds the more readily to rat albumin. Osorio and co-workers also found no evidence that clofibrate resembles salicylate and 30

dinitrophenol^ two drugs which have heen shown to displace thyroxine from

its "binding sites on plasma proteins in rats.

We next examined the inhibitory effect of 2, D-4 and L-^, and

p-chlorophenoxyacetic acid (37) on cholesterol biosynthesis vitro.

The inhibition of acetate-l-^^C and mevalonate-2-^^C incorporation into

nonsaponifiable material by acids 2, D-4 and L-4 and was determined

using rat liver homogenates. These results are shown in Tables 2 and 3,

respectively. The acid 2 is known to block sterol biosynthesis after

mevalonate^^ and before squalene with a minimal effect before mevalonate.

At a concentration of 1.5 nM acids 2, L-4, and 37 inhibits acetate in­

corporation about 30 per cent. The D-4 isomer exhibits less than one- i fourth the activity (p<0.02) of either 2, L-4, or 37 when acetate is the

precursor. The inhibition at the I .5 mM dose is slightly greater when

acetate rather than raevalonate is used, but with either precursor the

difference in activity between 2, L-4, and ^ is not significant. As

shown in Figure 3, dose-response curves further substantiate the greater

in vitro activity of acids 2, L-4, and 37- Within experimental error these three compounds exhibit essentially the same curve with approxl- lli mately 90 per cent inhibition of incorporation of mevalonate-2- C into nonsaponifiable material at a concentration of 9 niM. At this concentra­ tion of inhibition of incorporation is not more than 20 per cent.

We next examined the basic premise that when similar struc­ tures produce the same net biologic effect they likely do so by the 70 same mode of action. Combinations of either I .5 2 plus I .5 mM L-4 or 1 .5 mM 2 plus I .5 mM 37 give the same per cent inhibition of incorpo- lii ration of mevalonate-2- C into nonsaponifiable material as did 3*0 2 31

Tatle 2

Effect of Compounds 2, D-4 and h-k, and 37 Added ^ Vitro on the Incorporation of Acetate-l-l^CInto Nonsaponifiable Material of Rat Liver Homogenate

Compound ^ Inhibition of (1.5 mM l^C Incorporation^

2 3 0 ± 5^

L-U 33 ± 7

B-k 7 ± 5

M 2 7 ± 6

(a) Average value from five determinations. (b) Standard error of the average value.

Table 3

Effect of 2, D-4 and L-4 Added In Vitro on the Incorporation of Mevalonate-2-1^ Into Non­ saponifiable Material in Rat Liver Homogenate

Compound ^ Inhibition of (1.5 mM) ^^C Incorporation^

2 2k ± 3"b

L-^ 23 ± 3

B-k 7 ± 5

(a) Average value from five determinations, (h) Standard error of the average value. 100

M

H* H*

20

1.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0

Dose (bM)

Figure 3 Dose-Response Curves for the Per Cent Inhibition of Incorporation of Mevalonate-2-^^C Into Nonsaponifiable Material in Fortified Rat Liver Homogenate by Acids 2, L-(-)-4, D-(+)-4 and JT (Mean and extreme values for per cent inhibition.) U! ro 33 alone; i.e., no synergistic effects are observed. Had a synergistic

effect been found, this might have suggested two different sites in the overall patln/ay of sterolgenesis for the action of L-4 and 2 or 37.

Analysis by gas-liquid partition chromatography indicates that cholesterol accounts for approximately 85 per cent of the nonsaponifi­ able material extracted from rat liver homogenate preparations incubated with added drugs (controls) while squalene accounts for less than 2 per llj. cent of the total fraction. When mevalonate-2- C is added to the con­ trol rat liver homogenate preparations and the squalene and cholesterol are separated on acid-washed alumina, the ratio of radio-labeled choles­ terol to squalene is approximately 150:1. The purity of the cholesterol and squalene fractions eluted from the alumina column is substantiated by glpc.

Addition of 3*0 hM 2, L-4, or 3% to rat liver homogenate prep­ arations, followed by incubation and isolation of the radio-labeled squalene and cholesterol, shows, within experimental error, the same lJl per cent inhibition of incorporation of mevalonate-2- C into these com­ pounds as into nonsaponifiable material. These results with 2 agree with those obtained by others,^^^'*^ who found that various concentrations

(0 .25-2 .5 mM) of 2 inhibit cholesterol biosynthesis to the same extent as nonsaponifiable material both in liver and aortic preparations. Our results show that like 2, L-4 and 37 interfere with cholesterol biosyn­ thesis between mevalonate and squalene. The considerably lower drug activity of D-4 in vitro indicates that insertion of methyl in the wrong configuration might inhibit the binding of the parent structure 37^ yet apparently does not inhibit the association of 2 with the (presumed) common binding site(s) on the drug-sensitive enzymes. 34 l4 The inhibition of incorporation of mevalonate-2- C into non­ saponifiable material by acids 6c, Jc^, and l4 was also examined. The results are shown in Table 4.

Table 4

Effect of 6c, 7c, and l4 Added In Vitro on the Incorporation of Mevalonate-2- C Into Non­ saponifiable Material in Rat Liver Homogenate

Compound ^ Inhibition of (1.5 mM) Incorporation®'

6c 3 ± 3^

Jc 25 ± 3

l4 29 ± 0

(a) Average value from four determinations. (b) Standard error of the average value.

Dose-response curves for acids 6c, 7ç, and l4 are shown in Figure 4. Compounds 7ç and ]A both give dose-response curves similar to those for 2, L-4, and The dose-response curve for compound 6c shows a greatly diminished inhibitory effect. The high activity ex­ hibited by acids Jc and stimulates further interest in these com­ pounds. Resolution of Jc and 1À followed by testing ^ vitro of the optical isomers may supply further information concerning the mechanism of action of la. 100

80 1 o (D éO g40 O' c+ H< O 3 20

1.0 10.0 1.0 10.0 1.0 10.0 Dose (inM)

Figure 4 Dose-Response Curves for the Per Cent Inhibition of Incorporation of Mevalonate-2-^ C Into Nonsaponifiable Material in Fortified Rat Liver Homogenate (Mean and extreme values for per cent inhibition.)

(jO CHAFEER III. EXPERIMENTAL

Chemical

Melting points are corrected and are taken using a Thomas-

Hoover melting point apparatus. Optical rotatory dispersion and circular dichroism measurements are recorded with a Durrum-Jasco spectropolari- meter. Rotations at the sodium D line are taken with a Zeiss polari- meter. Infrared spectra are obtained with a Perkin-Elmer Model 257 grating spectrophotometer. The nmr spectra are recorded with a Varian

A-6OA nmr spectrometer at 60 MHz using TMS as an internal reference. Gas chromatographs are taken using an F and M model k02 gas chromatograph equipped with flame ionization detector and glass columns. Chemical analyses are determined hy Schwartzkopf Microanalytical Laboratory,

Woodside, N.Y., and by Clark Microanalytical Laboratory, Urbana, Illinois.

Ethyl g-(4-substituted phenoxy)-a-methylpropionates (l) are prepared by the method of Bargelline described by Julia and co-workers.

Both known and unknown compounds are described in Table 5.

g-(4-chlorophenoxy)-a-methylpropionic acid (2 ) is prepared according to the procedure of Bargelline described by Julian and co- workers,^^ mp 117 -118 °, lit.^“ 1 18 -119 °.

36 37 Table 5 Ethyl a- ( 4-Sub st itut ed-Phenoxy ) -a-Methylpropionat e s

______Bp, C(nnn)______Compound Formula Analyses Observed Literature

la C H 0 Cl C,H,C1 160-164°(30) 138°(10 )^^°

lb C H 0 C,H 175-180°(30) 139°(4)^^

Ic CH 0 C,H 150-154°(30) i 4o -i 42°(8)^^^

C H 0 C,H 138-142°(30) 1 60 -165°(7)^^^

le 0 H 0 N C,H,N 154-156°(4)

^Analyses were determined by Schwarzkopf Microanalytical Laboratory, Woodside, N.Y., and by Clark Microanalytical Laboratory, Urbana, Illinois, and were within 0.4^ of the calculated values.

Ethyl DL-g-(4-chlorophenoxy)propionate (3 ) is synthesized by

refluxing equimolar parts of potassium 4-chlorophenolate and ethyl a-

broraopropionate (3 3 ) in 2-butanone. 4-Chlorophenol (l^, 94 g, O .7 mole)

and ethyl 2 -bromopropionate (33, 122.5 e> 0*7 mole) are re fluxed for 48 hr

with 2 7 6 .4 g of potassium carbonate in 500 ml of 2-butanone. The reac­

tion mixture is cooled and poured into 50 ml of water. The ketone layer

is separated and the water layer is further extracted (ether), dried

(sodium sulfate), and filtered, and the combined ether and ketone layers

are concentrated under reduced pressure affording I50 g (89^) of 3 , bp

155-158° (2 0 ram), lit.^® bp 130 ° (0 .7 ram).

DL-g-( 4-Chlorophenoxy)propionic acid (4) is prepared from

150 g (0 .6 5 mole) of 3 by refluxing in lOJ^ aqueous sodium hydroxide with

stirring for 2 hr affording 121.9 g (665^) of DL acid. Recrystallization

from petroleum ether (60-8 0) affords white crystals, mp 103-104°, lit. mp 1 0 4 .5-1 0 5 .5°. 38

L-(- )-q-(lj-Chlorophenoxy)propionic acid (4) is obtained accord­ ing to the procedure of Mat ell. Finely powdered (-)-hrucine (1 6.T g,

0.042 mole) is suspended in 2 liters of boiling 20^ ethanol in water.

To the stirred suspension is added 20 g (O.l mole) of DL-4 in 50 ml of

IN sodium hydroxide and 50 ml of ethanol. The mixture is stirred with boiling water until a clear solution results. The solution is filtered and on cooling the salt crystallizes. Five additional recrystallizations from 20^ ethanol-water affords 10.6 g (44^) of brucine salt, [a]^^^^25.4°

(c 1 .9720, methanol). Utilizing twelve times these amounts 122 g of opti­ cally pure salt is obtained. The acid is liberated from 122 g of salt by acidification with 5^ sulfuric acid and extracted with ether. The ether is dried (sodium sulfate), filtered, and removed under reduced pressure affording after recrystallization from petroleum ether (60-80°), 2 8 .8 g

(70^) of L-(-)-4: mp 104-105°, lit.^^° mp 103-5-104.5°; [a]^^^^-34.95°

(c 5*0078, methanol), lit[a]^^^-40.1° (ethanol); ED (c 0.0242, methanol) (24-25°) [] 325-567°, []310 -803°, [*]30 Q-1320 °, [*]g^-2650°,

[ 4 .] 2 3 3 - 1 8 0 0 ° (sh), 0°, [*]g6y+1130°, [*]ggi^+ll80°, [4.]ggQ+990°,

0°, [4-]21 ^5-710 °, [*]gj^-l460°j CD (c 0.2420, methanol (24-25°),

[ 6 ) 3 0 0 0 ° , [ 6 ] 2 8 ^ - 2 6 2 0 ° , [ 6 ]20^-1750°, [01282-2990°, [8]gy3-2310°,

16]254-500 °, [6]22,0 -2500 °.

D-(+)-a-(4-Chlorophenoxy)propionic acid (4) is obtained using the method of Smith and co-workers 54 which was developed by them for resolving similar compounds. The combined mother liquors from the first two crystallizations of L-(-)-4 are acidified with 10^ sulfuric acid and extracted (ether). The ether is dried (sodium sulfate), filtered, and removed under reduced pressure affording 8 .6 g of crude acid. Resolution is accomplished utilizing (+)-yohimbine obtained from the hydrochloride 39 salt. (+)-Yohim'bine hydrochloride (8.4 g) is dissolved in O .5 liter of boiling water; the base precipitates with excess ammonium hydroxide, is filtered and washed with hot water. The moist base is suspended in

1 liter of 2.% ethanol; a mixture of crude acid (8.6 g, 0.04 mole) in

25 ml of IN sodium hydroxide and 25 ml of ethanol is added to the vig­ orously stirring suspension. The salt dissolves immediately and crystal­ lizes upon standing overnight at room temperature. Three recrystalliza­ tions of the yohimbine salt of D-(+)-4 from acetone affords white crystals;

[а]^^jj+64.8^ (c 2 .32 2 0 , methanol). The D-(+) acid is liberated from the salt as in L-(-)-4 above. Recrystallization from petroleum ether (60-80°) affords 4.0 g (46^) of white crystals: mp 104-105°; lit. mp IO3 .5-

104.7°; [a]^^j5+ 3 4.1 ° (c 3 -67 2 0, methanol), lit.^^° [a]^^jj+39-8° (ethanol);

ED (c 0 .0261 , methanol) (24-25°), [((>] 325+610 °, [<|)] 3^^0 +830°, [4»]^QQ+ll80°,

[

[0]25o 0°, [0]21 ^5+790°, [0]gi^Q+l490°; CD (c O.26IO, methanol) (24-25°),

[б )3 0 0 0 °; 10 )288+1 ^50°, [0)23^+2600°, [0 )2^0 +1560°, [0 )268+1450°,

[0 )253+460°, [0 )21 ^0 +2020 °.

Ethyl L-(-)-a-(4-chlorophenoxy)propionate 3 is prepared by re­ fluxing the L-(-) acid 4 (12 g, O.O6 mole) with 100 ml of ethanol con­ taining 4 ml of sulfuric acid for 24 hr. The solution is poured into

200 ml of water and extracted (ether). The ether is washed with 5^ sodium bicarbonate, dried (sodium sulfate), filtered, and removed under reduced pressure affording after distillation 8 .8 g (65^) of L-(-)-3 ester; bp 150 -152° (2 0 ram); [a)^5^_l|5.2° (c 5.0 1 2 0 , methanol), lit.5^^

[a]25^.5 2.7° (ethanol); ED (c 0.0302, methanol) (24-25°), [)325-680°, 4o

[*]300-1330°, [4.1289-21^60°, [*]281^-17W° (sh), [*1280-830°,

[* l2T4 0°, [4.12^0+680°, [*1266+720°, [*l26i+6W°, [*12530°, [*1250-530°,

[*1240-1^80°; CD (c 0 .3020 , methanol) (24-25°), [81300 -80°°; [0 ]2g^-235O°,

[0 1 2 8- 62 0 5 0°, [6 1 282- 2 7 5 0°, [01278- 2 25 0°, [0 1 2 7 5- 2 4 5 0°, [0125^^-900°,

[01245-1600 °.

Ethyl D-(+)-a-(4-chlorophenoxy)propionate (3 ) is prepared in

similar yields and by the same method used for the L-(-)-^ ester; bp l48-

152° (2 0 mm); [al^^^+46.5° (c 6.2328, methanol), lit.^®° [al^^^+5 3.5°

(ethanol); RD (c O.O296, methanol) (24-25°), [*1325+660°, [*1^QQ+12T0°,

[*1289+2510 °, [*1284+1890° (sh), [*1274 0 °, [*1255-770°, [*1255-770°,

[*lg53 0°, [*1240+1780°; CD (c O.296O, methanol) (24-25°), [*lgQQ+360°,

[*l288-"^®9°°> [*1284+^730°, [*1281 +2550°, [*1274+1990°, [*1255+460°, [*3240+^340°.

a-(4-Chlorophenoxy)-a-methylbutyric acid (lA) is obtained by

the dropwise addition of chloroform (135 ml) to a mixture of 4-chlorophenol

(1 2 , 1 2 8 .6 g, 1 .0 mole), finely powdered sodium hydroxide (200 g, 5*0 mole),

and 1 liter of 2-butanone. The reaction mixture is refluxed for 4 hr,

cooled, and the solvent removed under reduced pressure. The residue is

diluted with 1 liter of water and extracted with ether (discard ether).

The aqueous portion is acidified with 10% hydrocholoric acid, extracted

with ether (3 x 3OO ml) and the combined ether fractions are distilled under reduced pressure. The resulting residue is dissolved in 7% aqueous

sodium bicarbonate, precipitated with 10 % hydrochloric acid and extracted with ether (3 x 3OO ml). The combined ether extracts are dried (sodium

sulfate) and the solvent is removed under reduced pressure affording

5 8 .1 g (26%) of crude l4. Recrystallization from 10% acetic acid kl followed loy petroleum ether (60-80°) affords white crystals, mp 95-9^°^ lit. 95-96°. Ethyl l,4-henzodloxan-2-carhoxylate (^) is prepared according to the method described hy Koo and co-workers. To a solution of catechol

(15, 77 g, 0 .7 mole) in 300 ml of dry acetone is added 70 g of anhydrous potassium carbonate, then dropwise, with stirring and gentle refluxing, ethyl 2,3-dibromopropionate {}£, 50 g) is added. Another 70 g of potas­ sium carbonate and 30 g of 3^ is added similarly and repeated two more times, using altogether 280 g of potassium carbonate and 200 g (0 .8 mole) of 3^. Stirring and refluxing is continued for another I8 hr, while add­ ing dry acetone periodically to keep the reaction mixture fluid enough for stirring. The reaction mixture is then cooled, filtered and the residue washed with acetone. The acetone is concentrated to about 200 ml and diluted with 3 00 ml of cold water, precipitating an oil. The reac­ tion mixture is repeatedly extracted with ether, the ether extracts com­ bined and dried (sodium sulfate). The solvent is removed under reduced pressure and the residual liquid distilled affording 110 g (76^), bp 82°

(0 .0 5 mm) lit.^^ bp 105-107° (O.15 mm).

1 ,4-Benzodioxan-2-carboxyllc acid (6c) is prepared by gently heating a mixture of ethyl 1 ,4-benzodioxan-2-carboxylate {6a, 6 .2 g,

0 .0 3 mole) and 40 ml of 10 ^ aqueous sodium hydroxide on a steam bath for

30 minutes. The cooled reaction mixture was acidified with 10^ hydro­ chloric acid, chilled, and the precipitate filtered. The white solid is re crystallized from benzene-ligroin, affording 4.6 g (88^) of 6c, mp II8-

119 °, lit.^^ mp 1 19 -120 °. k2

^-ChloTO-g-'benzofurancar'boxylic acid (2 ) is prepared according 57 to a method of Kurdukar and Rao. A mixture of 5- chloro salicylaldéhyde

(1 0 0 .0 g, 0.64 mole, diethyl hromomalonate (1 0 0 .0 g, 0.42 mole, I8), anhydrous potassium carbonate (1 2 5 g) and 2-butanone (1 5 0 0 ml) are re­ fluxed for 7 hr. Following cooling, the reaction mixture is filtered and the solvent is distilled under reduced pressure. The resulting residue is refluxed in IO5S ethanolic potassium hydroxide (15OO ml) for O .5 hr using a steam bath. Following cooling, the alcohol is distilled under reduced pressure and 1 liter of water is added to the residue. The mix­ ture is acidified with 10 ^ sulfuric acid, filtered, dried (sodium sulfate) and the mixture recrystallized from ethyl acetate yielding 35*0 6 (57^) of

mp 259-260°, lit.^*^ 258°.

5-Chloro-2,3-dihydro-2-benzofurancarboxylic acid (7c) is pre- 73 pared according to a procedure described by Fredga. 5-Chloro-2-benzo- furancarboxylic acid (20, I5.O g, O.O8 mole) is added to a solution of sodium hydroxide (1 9 g) in 3OO ml of water. A sparingly soluble sodium salt separates. Sodium amalgam, prepared from 4.9 g of sodium and 195 g of mercury is added with stirring during 20 minutes. The suspended sodium salt goes into solution after 45 minutes. The solution is stirred for another 45 minutes and allowed to stand overnight. The mercury is sep­ arated and the solution filtered. Acidification of the aqueous solution with IO5& sulfuric acid precipitates the acid. Filtration and recrystal­ lization from petroleum ether (60-80°) affords 1 1 .8 g (75^) of (7ç), mp

114.5-115°' Nmr (Dg-acetone) ^ for the calculated ABX spectrum^^ shows

8 lines (AB, 2H, methylene protons) 6^ = 3«63, 6^ = 3*42, and 4 lines

(X, IH, methinyl proton), ôjj = 5*32 with = 1 6.8, = 10.74, and 43

*%X " cpsj (IH, cartoxyl proton) and a multiplet at 6.67-7-3 3 à_ (3H, aromatic protons . The aromatic region shows the typical ABC pattern for those protons which are also long range coupled to the methylene aliphatic

AB protons.

Anal. Calcd for CpKyClOg: C; 54.4; H, 3-6; Cl, 17-8. Found:

C, 54.7; H, 3-7; Cl, 17-5

Ethyl 5-chloro-2,3-dihydro-2-henzofurancarboxylate (^) is ob­ tained by refluxing 5-chloro-2 ,3-âihydro-2 -benzofurancarboxylic acid (7c),

35 ml of absolute ethanol, 20 ml of toluene and 0.04 ml of sulfuric acid for 6 hr using a Dean-Stark trap to remove water. The reaction mixture is cooled, then washed successively with 100 ml portions of water, satu­ rated sodium bicarbonate solution and water. The organic layer is sep­ arated and the aqueous portion extracted (ether). The combined organic layers are dried (sodium sulfate), filtered and the solvent removed under reduced pressure. The residual liquid is distilled under reduced pressure affording 5-5 g (96^) of bp 121 ° (0 .5 0 mm).

Anal. Calcd for Cjj^Hj^q^CIO^: C, 5 8.3 ; H, 4.9; Cl, 1 5.6. Found:

C, 5 8.0 ; H, 4.7; Cl, 1 5.5-

Attempted reaction of ethyl 5-chloro-2,3-dihydro-2-benzofuran- carboxylate (7a) with methyl iodide. - (l) Ethyl 5-chloro-2,3-dihydro-2- benzofurancarboxylate (ja, 7 -0 g, O.O3 mole) dissolved in 5 ml of dimethyl formamide is added dropwise at 0 ° in a nitrogen atmosphere with stirring to a suspension of sodium hydride (0 .0 3 mole, 0 .7 g) in 40 ml of dimethyl 74 formamide according to a procedure of Zaugg and co-workers. Methyl iodide (4.3 g, 0 .0 3 mole) is then added dropwise at 0 ° to the reaction mixture and stirred for 30 minutes. The reaction mixture is then stirred H at room temperature for 6 hr. Ethanol (2 ml) is added to the reaction mixture, followed hy 100 g of ice, and neutralization with 10 ^ hydro­ chloric acid. The solution is extracted with ether, dried (sodium sul­ fate), filtered, and the solvent removed under reduced pressure. Distil­ lation of the residual liquid affords ester (2) Starting material

7a is also obtained using dimetl^l sulfoxide in place of dimethyl forma­ mide as the solvent.

Attempted reaction of ethyl 5-chloro-2,3-dihydro-2-henzofuran- carboxylate (7a) with methyl iodide. - Ethyl 5-chloro-2,3-dihydro-2- benzofurancarboxylate (7a, 2 .3 g, 0 .0 1 mole), methyl iodide (4.3 g,

0.03 mole), sodium ethoxide (lO ml, 0.01 mole) and absolute ethanol

(5 ml) are heated in a sealed Pyrex tube at an oil bath temperature of

100° for 2 hr. The cooled contents of the tube are washed with water followed by ether. The aqueous portion is extracted (ether), the com­ bined ether portions dried (sodium sulfate), filtered and the solvent distilled under reduced pressure. Distillation of the resulting liquids affords ester 7a in quantitative yield.

Ethyl ^-chlorosalicylate (22) is prepared from $-chlorosalicylic acid (21 , 102 g, 0 .5 mole), absolute ethanol (3 2 0 ml) and 24 ml of sul- cO furic acid as described by McIntyre. A yield of 52 g (54^) is obtained, bp 120 ° (4,5 mm), lit.^® bp 124-125° ( U mm).

Ethyl a-(4-chloro-2-carboethoxyphenoxy)propionate (24) is pre­ pared by refluxing ethyl 5-chlorosalicylate (^, 10 g, 0 .0 5 mole), ethyl a-bromopropionate (^, 11 g, 0 .0 6 mole) and potassium carbonate (25 g) in 100 ml of dry acetone for 8 hr. The cooled reaction mixture is fil­ tered, the solvent removed under reduced pressure. Distillation of the resulting liquid affords l4.0 g (93^) of 24, bp 135° (0 .1 2 5 mm). Kmr h3. spectrum (neat, 6) shows a broad multiplet at 0.92-1.8 3 (9H, three methyl groups), hroad multiplet at 4.00-5*30 (5H, two methylene groups and methine proton) and multiplet at 6.78-7*83 (3H, three aromatic protons)*

Anal. Calcd for C, 55*9; H, 5*7; Cl, 11*8. Found:

C, 5 6.0 ; H, 5*7; Cl, 1 1 .9.

Attempted condensation of ethyl a - ( 4-chloro-2-carhoethoxyphenoxy). propionate (24) * - To a stirred solution of sodium ethoxide (0.02 mole,

15 ml) in 30 ml of anhydrous benzene is added dropwise ethyl a(4-chloro-2-

2 -carboethoxyphenoxy)propionate (24, 5*0 g, 0 .0 2 mole) dissolved in 10 ml of benzene. The reaction mixture is stirred for 4 hr at room temperature followed by refluxing for 1 hr. The solvent is removed under reduced pressure, the residue dissolved in ice-water, and neutralized with 10 ^ hydrochloric acid. The aqueous solution is extracted (ether), dried

(sodium sulfate), filtered and the solvent distilled under reduced pres­ sure. The resulting red solid gave a positive ferric chloride test for a phenol.

Attempted condensation of ethyl g - ( 4- chloro-2-carboethoxy- T5 phenoxy)propionate (24). - A procedure described by Conroy is followed in which ethyl a-(4-chloro-2-carboethoxyphenoxy)propionate (24, 7*2 g,

0 .0 3 mole) dissolved in 50 ml of anhydrous tetrahydrofuran is added to a suspension of sodium hydride (4*0 g, O.O3 mole) in 50 ml of tetrahydro­ furan with stirring under a nitrogen atmosphere. After 24 hr, the mix­ ture is re fluxed for 3 hr, cooled, and ethanol (5 ml) added. Ice and 4n hydrochloric acid are added, the aqueous portion extracted (ether), the ether extracts washed with saturated sodium bicarbonate solution, and dried (magnesium sulfate). Filtration, then removal of solvent under reduced pressure affords a solid giving a positive ferric chloride test* k6

Diethyl 2- ( 4-chlorophenoxy )-2-methylmalonate (27) is prepared

hy refluxing 4-chlorophenol (12 , 1 0 .0 g, 0 .0 8 mole), diethyl 2 -hromo-2 -

methylmalonate (^, 2 0 .2 g, 0 .0 8 mole), anhydrous potassium carbonate

(40 g) and l60 ml of dry acetone for 24 hr. The cooled reaction mixture

is filtered and the solvent removed under reduced pressure. The liquid

is dissolved in 200 ml of cold 55^ sodiimi hydroxide and extracted (ether),

dried (sodium sulfate), filtered and the solvent distilled under reduced

pressure. Distillation of the residual liquid affords 13*3 g (55^) of

27, bp 108° (0 .0 5 0 mm), llmr spectrum (DCCI3 , ^) shows triplet at 1.15

(*^CH3 ,CH2 " cps, 6h, 2CH3 ), a singlet at I .7 0 (3H, CHg), a quartet at

4.20 (4h, two methylene groups) and an aromatic Ag'Bg' spectrum at ^ =

6 .9 6 and = 7*20 with JAg'Bg' = 8 .8 cps.

Anal. Calcd for C14H17CIO5: C, 55.9; H, 5-7; Cl, 11.8. Found: C, 56.2; H, 5-7; Cl, 12.2.

Attempted cyclization of diethyl 2-(4-chlorophenoxy)-2-methyl- malonate (27) • - A procedure described by Fieser and Fieser^^ is employed

in which diethyl 2 - (4-chlorophenoxy)-2 -methylmalonate (2%) is added drop- wise with stirring to a melt of sodium chloride 6 g and aluminum chloride

(3 0 g) heated at 120°. The temperature is raised to 150° for 15 minutes,

cooled to room temperature and poured into an ice-hydrochloric acid bath.

The reaction mixture is extracted (ether), dried (sodium sulfate), fil­ tered and the solvent removed under reduced pressure. A black tar re­

sulted from which no small molecules could be isolated.

Attempted hydrolysis of diethyl 2-(4-chlorophenoxy)-2-methyl- malonate (27). - Diethyl 2-(4-chlorophenoxy)-2-methylmalonate (27, 5*0 g,

0 .0 2 mole) is stirred with a solution of potassium hydroxide (4 g. hi

0.04 mole) in 40 ml of absolute ethanol for 3*5 hr. The resulting sus­ pension is diluted with 100 ml of absolute ethanol and re fluxed for 0.5 hr.

The cooled reaction mixture is diluted with ice water, acidified with 10^ sulfuric acid, extracted (ether) and dried (sodium sulfate). Filtration and distillation of the solvent under reduced pressure affords a solid identified as 2-(4-chlorophenoxy)propionic acid (4).

Ethyl 4-chloro-2-carboethoxyphenoxyacetate (^) is prepared by condensation of ethyl 5-chlorosalicylate (22, 20.1 g, 0.1 mole) with ethyl 2-bromoacetate (2 8, l6.7 0.1 mole) in 200 ml of dry acetone using potassium carbonate (50 g). The reaction mixture is refluxed 10 hr, cooled, filtered and the solvent removed under reduced pressure. 27*0 g

(9 4.1 ^) of a white solid results which is carried on to the next step with further purification.^^

5-Chloro-2-carboetho):y-3(2H)-benzofuranone (30 ) is prepared by the method described by Schroeder and co-workers. 5-Chloro-2-carbo- ethoxy-3(2H)-benzofuranone (30j 10.0 g, O.O3 mole) dissolved in 50 ml of dry benzene is added dropwise with stirring to sodium ethoxide (1 5 ml,

0.03 mole) in 250 ml of dry benzene, and the reaction mixture is refluxed for 4 hr. After cooling, the reaction mixture is diluted with water, acidified with 10^ hydrochloric acid and extracted (ether). The ether is removed under reduced pressure and the resulting solid recrystallized from ethanol, affording 6 .2 g (7 6.5^) of rap 1 2 8-129°, lit.^^ rap 129-130 °.

Attempted méthylation of 5-chloro-2-carboethoxy-3(2H)-benzo- furanone (3 0 ) using sodium ethoxide. - 5-Chloro-2-carboethoxy-3(2H)- benzofuranone (3 0 , 2 .5 g, 0 .0 1 mole) is dissolved in 100 ml of absolute ethanol, and methyl iodide (28.4 g, 0.2 mole) added. Sodium ethoxide 48

(25 ml, 0.01 mole) is added dropwise with stirring and the reaction mix­ ture refluxed for 2 hr. Ice water is added to the reaction mixture, and neutralization carrier out with 10^ sulfuric acid. The reaction mixture is extracted (ether), washed with water and dried (sodium sulfate). Fil­ tration and removal of solvent under reduced pressure affords starting material 30»

Ethyl 5-chloro-3-methoxy-2-henzofurancarhoxylate (3 1 ) is ob­ tained using sodium hydride in dimethyl sulfoxide. 5-Chloro-2 -carho- ethoxy-3 (2H )-benzofuranone (3 0 , 1 0 .0 g, 0.04 mole) dissolved in 200 ml of absolute ethanol for 2.5 hr. The cooled reaction mixture is diluted with water and extracted with ether. The combined ether extracts are dried, filtered and the solvent removed under reduced pressure affording

5 .9 g (80^) of a solid identified as 3I.

5-Chloro-2-methyl-2-carboethoxy-3(2H)-benzofuranone 25 is pre­ pared by heating 5-chloro-2-carboethoxy-3(2H)-benzofuranone (^, 2.0 g,

0 .0 0 8 mole), sodium ethoxide (lO ml, O.OO8 mole), methyl iodide (3»0 g,

0.021 mole) and absolute ethanol (lO ml) in a sealed Fyrex tube at an oil-bath temperature of 100 for 2 hr. The cooled solution is poured into a separatory funnel and the tube washed with water and ether. Salt water

(100 ml) is added to the reaction mixture and the aqueous phase extracted

(ether) until all color has disappeared from the aqueous phase. The com­ bined ether extracts are washed with three 100 ml portions of cold 5^ sodium hydroxide, 100 ml of water, and 100 ml of saturated sodium chloride solution, followed by drying (magnesium sulfate). The ether solution is filtered and the solvent removed under reduced pressure. Distillation of the resulting yellow liquid affords 1 .1 g (50^) of 2 5, bp 112-114° 49 (0-55 rm) » The distillate immediately solidifies upon standing at room temperature and is re crystallized from 90^ ethanol, mp h6-kj.^°. Nmr spectrnm (DCCl^, shows a triplet at 1.23 (3H, methyl group of ethyl ester, cps), a sharp singlet at 1.73 (3H, C-methyl group), a quartet at 4.22 (2H, methylene group of ester) and a multiplet at

6.92-T-T5 (3H, three aromatic protons).

Anal. Calcd for CigHpiClOi^.: C, $6.6; H, 4.4; Cl, 13-9-

Found: C, $6.8; H, 4.5; Cl, 13-9.

Attempted reaction of 5-chloro-2-methyl-2-carhoethoxy-3(2H)- henzofuranone (25) with ethanedithiol. - A procedure described by

Sondheimer and Rosenthal*^^ is followed in which boron trifluoride etherate (0.3 ml) is added to a mixture of 5-chloro-2-methyl-2-carbo- ethoxy-3(2H)-benzofuranone (25, 1.0 g, 0.004 mole) and ethanedithiol

(0.6 ml), with ice cooling and the mixture is then allowed to stand at room temperature for 10 minutes. Methanol (3-0 ml) is added, the mixture is cooled in ice and scratched. No crystals resulted. Further cooling and scratching afforded no crystals.

Ethyl 5-chloro-3-(p-toluenesulfonylhydrazone)-2-methyl-2-benzo- furancarboxylate (^) is prepared by refluxing 5-chloro-2-methyl-2- carboethoxy-3(2H)-benzofuranone (25, 1.5 S, O.OO6 mole), p-toluenesul- fonylhydrazide (3I, 1.1 g, O.OO6 mole), 3 drops of concentrated hydro­ chloric acid and 30 ml of absolute ethanol for 4 hr. The cooled reaction mixture is diluted with water and extracted (ether). The ether layer is washed with 50 ml portions of water, saturated sodium bicarbonate solution, and water, and dried (sodium sulfate). The solvent is distilled under re­ duced pressure affording 2.5 6 of a crude solid which upon recrystallization 50 from benzene affords 1.7 g (67%) of J2, mp 17^-175° • Nmr spectrum (Dg- dimethyl sulfoxide, shows triplet at I.I6 (3H, methyl group of ethyl ester, = 7-0 cps), a sharp singlet at I.7I (3H, methyl group bonded to the furan ring), a sharp singlet at 2.k2 (3H, methyl group attached to aromatic ring), a quartet at 4.20 (2H, methlene group), and a multiplet at 7*00 -8 .1 3 (8h, aromatic protons).

Anal. Calcd for C22 HigClN20 $S: C, 54*0; H, 4.5; Cl, 8.4;

N, 6.6; S, 7.6. Found: C, 54.1; H, 4.6; 01, 8.3 ; N, 6.8; S, 7*6.

Attempted reduction of ethyl 5-chloro-3-(p-toluene-sulfonyl- hydrazone)-2 -methyl-2 -benzofurancarboxylate (32 ) with sodium borohydrlde. - 77 A procedure described by Caglioti and Gras se Hi is followed in which ethyl 5-chloro-3-(p-toluenesulfonylhydrazone)-2 -methyl-2 -benzofuran- carboxylate (^), I .3 g, O.OO3 mole), sodium borohydride (0.044 g, I .5 equivalents and dioxane (5 0 ml) are refluxed 8 hr. The cooled reaction mixture is poured into ice and hydrochloric acid, and extracted (ether).

The dried (sodium sulfate) ether layer is filtered and the solvent dis­ tilled under reduced pressure affording a red oil which is not the de­ sired product 7b and has not been characterized.

Attempted reduction of 5-chloro-2-methyl-2-carbo-ethoxy-3( 2H )- benzofuranone (25) employing Clemmenson conditions. - A mixture of mossy zinc (2 0 g), mercuric chloride (5 g), concentrated hydrochloric acid

(2 ml), and 30 ml of water is vigorously stirred for 10 minutes, the aqueous layer decanted off and 30 ml portions of water and concentrated hydrochloric acid are added to the amalgamated zinc residue. 5-Chloro-

2-methyl-2 -carboethoxy-3(2H)-benzofuranone (2 5) dissolved in 10 ml of glacial acetic acid is added to the amalgamated zinc preparation and 51 the reaction mixture stirred overnight at room temperature . The reaction

mixture is gently warmed on a steam hath for 1 hr, cooled, extracted

(ether) and the ether layer washed with water followed hy saturated

sodium hicarhonate solution. The aqueous portion is acidified to deter­

mine if any acidic products are obtained from the reaction. The ether

layer is dried (sodium sulfate), filtered and the solvent is removed

under reduced pressure affording a white solid which is recrystallized

from petroleum ether (60-80°). The desired product Jh is not obtained

and the reaction product has not been characterized.

5-Chloro -2,3- dihydro - 2 -hydr oxyme t hylhenz ofuran (3 3) is prepared

by the dropwise addition of 5-chloro-2 ,3-dihydro-2 -benzofurancarboxylic

acid (7c, 4.^ g, 0 .0 2 mole) dissolved in 30 ml of ether to a stirred sus­

pension of lithium aluminum hydride (1.7 g, 0.04 mole). After stirring

overnight, the reaction mixture is cooled to 0 ° and 20 ml of water added

dropv;ise followed by 60 ml of 10^ sulfuric acid. The reaction mixture is

extracted (ether), the ether layer washed with water, saturated sodium bicarbonate solution and water, and dried (sodium sulfate). Filtration

and removal of the solvent under reduced pressure affords a viscous liquid

which following distillation affords 3*5 g (70^) of 33^ bp 107-109°

(-0.025 mm). Upon standing, the liquid solidifies to a solid, mp 4l-42.5°-

Kmr spectrum (carbon tetrachloride, ^) shows a singlet at 2 .7 1 (IH,

hydroxyl proton), a multiplet centered at 3-0^ (2H, two benzylic protons),

a multiplet at 3 *50-3*77 (2H, methylene group bonded to hydroxyl), a broad

multiplet at 4.47-5-13 (iH, methine proton), and a multiplet at 6.47-7.12

(3H , aromatic proton s).

Anal. Calcd for C^^ClOp: C, 5 8.7j H, 4.9; Cl, 19.2. Found:

C, 59-2; H, 5.I; Cl, 1 8.6. 52

5-Chloro-2; 3-d^hydro-2-hydroxymethylbenzofuran methane sulfonate

(3 4) is prepared by the dropwise addition of methanesulfonyl chloride

(1 .9 g; 0 .0 1 7 mole) in O.J ml of pyridine to a stirred solution of 5- chloro-2 ,3-dibydro-2 -hydroxymethylbenzofuran (2 3 , 3*0 g, 0 .0 1 7 mole) in

1 .7 ml of pyridine. The solution turns to a precipitate and is allowed to stand overnight. The solid is diluted with water and extracted

(ether). The dried (sodium sulfate) ether extract is filtered and the solvent is removed under reduced pressure yielding a white solid, which following recrystallization from ethanol, affords 2 .6 g (59^) of 3ji, mp

87-88°. Nmr spectrum (Dg-diraethyl sulfoxide, ^) shows a multiplet at

2 .75-3*67 (5H, two benzylic protons and methyl group attached to sulfur), a multiplet at 5*97-6.33 (2H, methylene group attached to methanesul- fonate group), a broad multiplet at 4.83-5*^0 (iH, methine proton).

Anal. Calcd for CioHnC10l|S: C, 45.7; H, 4.2; Cl, 13*5; S,

12.2. Found: C, 45.8; H, 4.4; Cl, 13*4; S, 12.4.

Attempted reaction of 5-chloro-2,3-dihydro-2-hydroxymethylben- z ofuran methane sulfonate (3^) with cyanide ion. - To a solution of 5- chloro-2 ,3-dihyd ro -2 -hydroxymethylbenz ofuran methane sulfonate (3^, 0 .8 g,

0 .0 0 3 mole) in 20 ml of dimethyl formaraide is added a solution of potas­ sium iodide (O.OO6 g) and potassium cyanide (0.254 g, 0.004 mole) dis­ solved in 2 .0 ml of water, according to a procedure described by

Pelizzour and Jommi.^^ The solution is heated at 80° for 8 hr. The cooled solution is extracted (ether), the ether dried (sodium sulfate), filtered and the solvent removed under reduced pressure. A black oil results which could not be recrystallized and would not distill. The infrared spectrum (chloroform) exhibits no triple bond absorption in the

2260-2240 cm~^ region. 53 g-Chloro-2,3-diliydro-2-chloromethyl'benzofuran (35) is synthe-

sized according to a procedure of Brooks and Snyder. Thionyl chloride

(3 .4 g; 0 .0 2 9 mole) is added dropwise at 0° to a rapidly stirring reac­ tion mixture of ^-chloro-2;3-dihydro-2-hydroxymethylhenzofuran {3^, 5»0 g,

0 .0 2 7 mole) and pyridine (2.4 g, 0.027 mole). The reaction mixture is allowed to stir for 4 hr at room temperature. The dark hrovn suspension is diluted with water and extracted with ether. The dried (sodium sul­ fate) ether extract is filtered, the solvent removed under reduced pres­

sure and the resulting liquid distilled affording 1.7 g (30^) of hp

86° (0 .0 2 5 mm). The liquid crystallized upon standing and is recrystal­ lized from petroleum ether (60-80°), mp 54-55°* Infrared spectrum (chlor­ oform) exhibits no hydroxyl absorption in the 3200-3700 cm"^ region. Nmr spectrum (carbon tetrachloride, ^) shows a broad multiplet at 2 .77-3*88

(4h, two methylene groups), a broad multiplet at 4.65-5*20 (IH, a methine proton) and a multiplet at 6.49-7*17 (3H, three aromatic protons).

Anal. Calcd for CgHgClgO: C, 53*2; H, 4.0; 01, 34.9* Found:

C, 53*5; H, 4.0; Cl, 34.5* Attempted preparation of grignard reagent of 5-chloro-2,3- dihydro-2-chloromethylbenzofuran (35) * - Using equipment which is oven- dried, a crystal of iodine is added to a flask containing magnesium (l.O g,

0.04 mole) and 50 ml of purified tetrahydrofuran. With stirring, a 10-ml portion of a solution of 5-chloro-2 ,3-dihydro-2 -chloromethylbenzofuran

(35, 4.0 g, 0 .0 2 mole) and methyl iodide (2 .8 g, 0 .0 2 mole) in 50 ml of purified tetrahydrofuran is added under a nitrogen atmosphere. When no reaction occurs, the reaction mixture is refluxed using a water bath.

The rest of the solution is added but no reaction occurs; starting material is recovered. 3h

5-Chloro-2 ,3-âihydro-2-'benzofuranoyl chloride (3 6) is obtained

by refluxing 5-chloro-2 _,3 -

0 .0 3 mole) and thionyl chloride (6 ml) in ^0 ml of dry benzene for 6 hr.

The reaction mixture is cooled and the solvent removed under reduced pres­

sure. Distillation of the residual liquid affords 3*8 g (58^) of 3^, bp

110-112° (0 .5 mm). Nmr spectrum (deuterochloroform, ^) shows a multiplet

at 3 .OO-3 .9O (2H, two benzylic protons), a multiplet at 5*15-5-53 (IH,

methine proton) and a multiplet at 6.98-T.2 3 (3H; three aromatic protons).

Anal. Calcd for C^gClgO: 0, 49*8; H, 2.8; 01, 32.7» Found:

c, !^9.9j H, 2.9; Cl, 3 2 .7.

Attempted Amdt-Eistert synthesis employing 5-chloro-2,3-

dihydro-2-benzofuranoyl chloride ( ^ ) . - A procedure described by Misti 79 and co-workers is followed in which 5-chloro-2 ,3-dihydro-2 -benzofuranoyl

chloride (3 6, 5*2 g, 0.024 mole) dissolved in 50 ml of ether is added to

0 ° to 400 ml of an ethereal solution of freshly distilled diazomethane / (prepared from 20.6 g of nitrosomethylurea) with stirring. The reaction

mixture is then allowed to stand at room temperature for 17 hr. The sol­

vent is evaporated under a nitrogen stream leaving a yellow oil which is

not purified. The oil is dissolved in 60 ml of absolute ethanol and

stirred at 50° with silver oxide (1.2 g) for 8 hr. Filtration of the

cooled reaction mixture followed by removal of the solvent under reduced

pressure affords a liquid residue which is distilled, bp 120 ° (0 .2 0 0 ram).

The liquid solidified following distillation, mp 93~9^°* The solid is

recrystallized from petroleum, ether (60-80°). The distilled product is

not the desired compound and remains to be characterized. 55 Attempted Amdt-Eistert synthesis with 5-chloro-2; 3-dihydro-2-

henzofuranoyl chloride ( ^ ) . - A modification of the Arndt-Eistert syn-

thesis proposed hy Newman and Beal was next investigated. A solution of

2 .0 g of silver henzoate in 7 ml of triethylamine is added to a solution

of the oil (6 .6 g) obtained in the above experiment in 35 ml of absolute

methanol. A rapid evolution of gas ensues and the reaction mixture turns

dark black. Characterization of the product of this reaction will be dis­

cussed in a future communication.

Biological

In vivo activity is determined using diets containing 0.15,

0.20, and 0.50% ester. The compounds are dissolved in 500 ml of ether \ and the solution is thoroughly mixed with 2.5 kg of standard Purina rat

chow so as to impregnate the pellets. The ether evaporated on standing

for 12 hr. Six rats (Swiss Webster) are used as a control and adminis-

, tered a diet free of drug. Another six rats are used for each drug at

each dosage level. The rats are weighed daily. At the end of 12 days of

feeding, the control as well as the experimental group consumed 2 .5 kg of

food per six rats. The rats are anesthetized with ether, the aorta is

surgically exposed, and the blood is collected from analysis. Serum cho-

lesterol is determined by the method of Abell and co-workers.71

In vitro activity is studied in livers obtained from healthy

male Wister rats (200-250 g). Liver homogenates are prepared by Bucher's TP method and debris is removed by slow centrifugation (300 g, 2 .5 min).

One liver (lO g) yields approximately 20 ml of supernatant. Each incuba­ it tion flask contains 2 ml of supernatant, 2.5 |uCi of acetate-1- C, 0.1 ml 56 8o of stock drug solutions, and cofactors with added sodium citrate (final concentration 2 mM) made up to 4-.0 ml with O.IM phosphate buffer, pH

In the mevalonate-2-^^C studies, when drug concentrations are increased the volume of drug solution is likewise increased (due to limited drug solubility). Consequently, the volume of rat liver homogenate employed is decreased to I .5 ml. Concentrations of a U cofactors remain the same and the final volume is similarly made up to 4.0 ml with O.IM phosphate buffer. After 1 hr of incubation in air with slow shaking at 37° the reaction is stopped by adding 3-0 ml of lyjo potassium hydroxide in $0% ethanol followed by heating at 65.70° in a water bath for 10 minutes.

The partially saponified reaction rainture is transferred to a 20-ml glass-stoppered centrifuge tube and the flask is rinsed with 3 .0 ml of the alcoholic potassium hydroxide solution which is added to the contents of the centrifuge tube. Saponification is completed by heating the tightly stoppered tube in a water bath (75-80°) for 1 hr.

The saponified mixtures are extracted three times with 5-0-ml portions of petroleum ether (60-80°). The combined extracts are diluted to 2 5 .0 ml and dried (sodium sulfate). Five ml of the dried petroleum ether extract is diluted with 10.0 ml of PPO (2 ,5-diphenyloxazole) solu­ tion (0 .4^ PPO in toluene-959^ ethanol, 70:30 v/v) in standard counting vials. The radioactivity is determined with a Packard Tri-Carb liquid scintillation counter. A sufficient number of counts is taken to reduce the statistical error of counting to less than yjo. In each run, as a further check on the extraction procedure (and as a measure of possible contamination), the contents of one incubation flask is treated with potassium hydroxide solution prior to incubation; the radioactivity in 57 the petroleum ether extract derived from these alkali pretreated incuba­ tions is always found to he negligible.

Gas-liquid partition chromatography of the nonsaponifiable ma­ terial (1 5 ml) is concentrated to dryness (water hath, 50°) under nitro­ gen. The residue is dissolved in 0-5 ml of petroleum ether and gas chromatographed on 10^ silicone gum rubber (SE-3 0 ) on Chromosorb W (8OO-

100 mesh), if- ft x 0 .2 5 in. glass column with the column temperature 2 h0 °, detector temperature 285°, injection port temperature 355°, inlet pres­ sure of 2.8 kg/cra^, and carrier gas (He) flow rate 50 ml/minutej this gave a retention time for squalene of I3 .O minutes and for cholesterol,

24.4 minutes.

Separation of the nonsaponifiable extract into squalene and 81 cholesterol is accomplished by the method of Langdon and Bloch. The petroleum ether extract (1 5 ml) is concentrated to dryness in a water bath (50°) under nitrogen. The residue is chromatographed on acid- washed alumina using petroleum ether (60-80°). The squalene fraction is checked for purity by glpc. Sterols are then eluted with acetone- ether (l:l v/v). The cholesterol fraction is similarly checked for purity using glpc. Squalene- and cholesterol-containing eluates are concentrated to dryness in a water bath (50°) under nitrogen in standard counting vials. PPO solution (10 ml) is added and the radioactivity is determined with a Packard Tri-Carb scintillation counter. CHA.PTER IV. SUMMARY

This thesis is concerned with the synthesis and biological

evaluation of a series of ethyl oc-(if-substituted-phenoxy)-a-methylpro-

pionates and analogs in order to investigate the mechanism of action of

ethyl a - ( 4- chlorophenoxy ) -a-methylpropi onat e (Atrcanid S), a hypocholes­

térolémie agent. Substituents are varied in the 4-position to determine

their effect on hypocholesterolemic activity. The 4-chloro and hydrogen

compounds exhibit significant hypocholesterolemic activity in vivo in

rats.

The desmethyl analog, ethyl a-(4-chlorophenoxy)propionate is

synthesized and resolved into optical isomers. The two stereoisomers

are desired for biological evaluation to determine minimum structural

/ requirements for activity both ^ vivo and ^ vitro. The L-enantiomer

is found to exhibit significant hypocholesterolemic activity in vivo.

The effect of these stereoisomers on the incorporation of acetate-1- l4 C l4 and mevalonate-2- C into nonsaponifiable material, squalene and cho­

lesterol is discussed. The L-isomer is found to interfere with choles­

terol biosynthesis between mevalonate and squalene in rats. Certain

albumin binding studies involving these two stereoisomers are discussed.

These studies do not support the hypothesis that Atromid-S acts to lower

cholesterol levels by displacing thyroxine from its binding site(s) on

rat albumin.

58 59 The synthesis of certain henzodioxane and henzofuran analogs of Atromid-S is also discussed.

The attempted synthesis of 5-chloro-2j3-dihydro-2-henzofuran- acetic acid, an analog of L-mevalonic acid as well as Atromid-S, is discussed. CHAPTER V. REFERENCES

(1) G. L. Duff and G. G. McMillan, Am. J. Med.,11, 92 (1951).

(2) H, D. Moon and J. F. Rinehart, Circulation, 481 (1952).

(3) G. S. Getz, D. Vesselinovitch and R. W. Wissler, Am. J. Med., 657 (1969).

(4) J. T. Kassir, J. Fac. Med., Baghdad, JIO, 109 ^1968).

(5) W. Schrade, R. Biegler and E. Bob le, Schweiz. Med. Waschr., 89^, 117 (1959).

(6) C. S. McArthur, Biochem. J., 36, 559 (1942).

(7) F . B. Luddy, R. A. Barford, R. W. Riemenschenider and J. D. Evans, Arch. Biochem. Biophys., 817 (1959).

(8) D. B. Zilversmit, Ann. N. Y. Acad. Soi., 149, 710 (1968).

(9) A. B. Gutman, Am. J. Med., 14, 1 (1953).

(10) R. Gubner and H. E. Ungerleider. Am. Heart J., 436 (1959).

(11) (a) R. Pick, J. Stamler, S. Rodbard and L. M. Katz, Circulation, 6, 269 (1952); (b) G. L. Curran and D. L. Azarnoff, Arch. Int. Med., 101, 685 (1958).

(12) (a) M. F. Oliver and G. S. Boyd, Circulation, ]^, 82 (1956); (b) R. M. French, A. L. Cornish and J. B. Selley, J. Chron. Dis., 16, 236 (1963); (c) P. Starr, Arch. Int. Med., 101, 722 (1958); (d) P. Starr, J. Clin. Endocr., 20, 116 (1960); (e) J. Marmora ton, F. J. Moore, 0. T. Kuzma and J. Weiner, Proc. Soc. Exp. Biol. Med., 105, 618 (1960).

(13) l-[ (4-Diethylaminethoxy)phenyl]-l-(£-tolyl)-2-(£-chlorophenol) ethanol.

(14) Trans-l,4-his(2-chlorob enzy laminome thy 1 ) cy c 1 ohexane i^^Wdziochloride.

(15) Clofibrate, Chlorpenisate, ICI-28257- n .

(16) J. M. Thorp, Lancet, _1, 1323 (1962). 6o é l (17) (a) M. F. Oliver, J, Atheroscler. Res., 2» 427 (1963); (b) L. Hellman, B. Zumoff, G. Kessler, E. Kara, I. L. Rubbin and R. S. Rosenfeld, ibid., 2> 454 (1963); (c) R. P. Howard, P. Alaupovic, 0. J. Brusco and R. H. Furman, ibid. , 3, 482 (1963); (d) D. Berkowitz, ibid., 3, 538 (1963).

(18) (a) Symposium on Atromid, 2» 341 (1963); (b) P. J. Scott and P. J. Hurley, J. Atheroscler. Res., 2, 25 (1969); (c) M. M. Best and C. H. Duncan, J. Lab. Clin. Med., 64, 634 (1964); (d) D. S. Platt and J. M. Thorp, Biochem. Pharmacol. , L5, 915 (1966); (e) P. Weiss and J. R. Bianchine, Amer. J. Med. Sci., 258, 275 (1969); (f) L. S. Villadolid, A. I. Iledan, E. 0. Bacani and L. Velarde, Acta Med. Philipp., 2> 164 (1969).

(19) (a) J. Engstrom, Acta Med. Scand., 180, 519 (1966); (b) L. V. Baranova, Kardiologiya, 2, 65 (1965); (c) V. Kallio and L. B. Sourander, Gerontol. Clin., 8, 349 (1966); (d) R. G. Gould, E. A. Swyryd, B. J. Coan and D, R. Avoy, J. Atheroscler, Res., 6, 555 (1966); (e) E. D. Docking, R. Chakrabarti, J. Evans and C. R. Fearnley, ibid., %, 121 (1967); (f) V. S. Zadionchenko, Sov. Med., 22, 77 (1967); (g) E. S. Argaln, M. D. Bogdonoff and C. Cain, Arch. Internal Med., 119, 80 (1967); (h) R. W. Robinson and R. J. LeBeau, Amer. J. Med. Sci,, 253, 106 (1967) ; (i) J. W. Davis, S, J. Wilson and J. R. McField, ibid. , 252, 697 (1966); (j) J. R. O'Brien and J. B. Heywood, Thromb. Diath. Haemorrhag., 16, 768 (1966); (k) A. Herrernan, N. J. Hickey, R. Mulcahy and R. MacFarlane, Irish J. Med. Sci., 490, 423 (1966); (1) G. Frandoli, A. Gibelli and P. Giarola, Clin. Therap., 38, 27 (1966); (m) E. Giety, Med. Klin. (Munich), 336 (1967).

/ (20) Y. H. Chang, R. Pinson, Jr. and M. H. Malone, Biochem. Pharmacol., 16, 2053 (1967).

(21) (a) J. M. Thorp and W. S. Waring, Nature, 194, 948 (1963); (b) D. R. Avoy, E. A. Swyryd and R. G. Gould, J. Lipid Res., 369 (1965); (c) P. J. Kestel, E. Z. Hirsch and E. A. Couzens, J. Clin. Invest., 891 (1965) ; (d) D. L. Azarnoff, D. R. Tucker and G. A. Barr, Metabolism, 14, 959 (1965) ; (e) D. L. Azarnoff and D. R. Tucker, Fed. Proc., 22, 552 (1964); (f) S. W. Teal and W. Gamble, Biochem. Pharmacol., 14, 896 (1965).

(22) J. P. Peters and E. B. Mann, J. Clin. Invest., 22, 715 (1943).

(23) R. P. Rivers and J. R. Tate, in "The Thyroid Hormones," W. R. Trotter, Ed., Pergamon Press, New York, N. Y., 1959, pp. 79.

(24) T. A. Miettinen, J. Lab. Clin. Med., 71, 537 (1968).

(25) R. L. Searcy, D. A. Hungerford and E.M.Y. Low, Current Therap. Res., 10, 177 (1968). 62 (26) (a) R. H. Roseaman, M. Friedman and S. 0. Byers, Circulation, _5, 589 (1952); (b) R. H. Rosenman, M. Friedman and S. 0. Byers, J. Clin. Endocrinol., 12, 1287 (1952).

(27) (a) C. Eriksson, Acta. Chem. Scandinav., 10, 1055 (1956); (b) C. Eriksson, Proc. Soc. Exp. Biol. Med., 94, 582 (1957).

(28) D. Kritchevsky, Metabolism, 9, 984 (1960).

(29) C. D. Eskelson, Life Sciences, %, 467 (1968).

(30) S. B. Weiss and N. Marx, J. Biol. Chem., 213.349 (1955).

(31) C, H. Duncan and M. M. Best, Endocrinology, 169 (1958).

(32) (a) J. Robbins and J.‘ E. Rail, Recent Prog. Horm. Res. , JL3, 161 (1957) ; (b) J. Robbins and J. E. Rail, Physiol. Rev. , 415 (1960).

(33) (a) J. M. Thorp, J. Atheroscler. Res., 2, 351 (1963); (b) J. M. Thorp, in "The Control of Lipid Metabolism," J. K. Grant, Ed., Biochemical Society Symposia, Oxford, Academic Press, Inc., New York, N. Y., 1963, pp. 163.

(34) (a) K. Hellstrom and J. Sjovall, J. Atheroscler. Res., %, 205 (1961); (b) K. Hellstrom and S. Lindstedt, J. Lab. Clin. Med., 63, 666 (1964).

(35) A. S. Truswell, W. D. Mitchell and S. McVeigh, J. Atheroscler. Res., 6, 591 (1966).

(36) C. Osorio, K. W. Walton, C. H. Bro;

(37) W. W. Westerfield, D. A. Richert and W. R. Ruegamer, ibid., 17, 1003 (1968).

(38) R. Hess, W. Staubli and W. Riess, Nature (London), 208, 856 (1965).

(39) W. R. Ruegamer, G. H. Newman, D. A. Richert and W. W. Westerfeld, Endocrinology, 77^, 707 (1965).

(40) W. R. Ruegamer, N. T. Ryan, D. A. Richert and W. W. Westerfeld, Biochem. Pharmacol., 18, 613 (1969).

(41) A. L. Tarentino, D. A. Richert and W. W. Westerfeld, Biochem. Biophys. Acta, 124, 295 (1966). 63

(42) (a) E. H. Strisower, A, V. Nichols, F. T. Lindgren and L. Smith, J. Lab. Clin. Med., 6^, 748 (1965); (b) C. H. Duncan, M. M. Best and A. Despopoulos, Circulation, suppl. 7 (1964); (c) N. Spritz, Diabetes, 14, 467 (1965).

(43) H. R. Mahler and E. H. Cordes, in "Biological Chemistry," Harper and Row, Publishers, Inc., 1966, pp. 441.

(44) (a) R. 0. Brady, J. Biol. Chem., 193, 145 (1951); (b) E. Caspi, G. Rosenfeld and R. I. Dorfman, J. Org. Chem., 2A, 814 (1956) ; (c) D. Stone and 0. Hechter, Arch. Biochem. Biophys., 457 (1954).

(45) C. Berry, A. Moxham, E. Smith, A. E. Kellie and J.D.N. Nabarro, J. Atheroscler. Res., 2» 380 (1963).

(46) D. H. Huffman and D. L. Azarnoff, Steroids, £, 41 (1967).

(47) J. C. Hoak, W. E. Connor, M. L. Armstrong and E. D. Warner, J. Lab. Invest., 19, 370 (1968).

(48) R. G. Gould, E. A. Swyrd, D. Avoy and B. Coan, in "Progress in Biochemical Pharmacology," Karger, Basel, Vol. 1967, pp. 345.

(49) M. R. Walsh, S. W. Teal and W. Gamble, Arch. Biochem. Biophys., 130, 7 (1969).

(50) (a) W.G.M. Jones, J. M. Thorp and W. S. Waring, British Patent 860, 303 (1961); (b) J. M. Thorp, British Patent 898, 596 (1962).

(51) (a) M. Julia, M. Baillarge and G. Tschernoff, Bull. Soc. Chim. France, 776 (1956); (b) G. Bargellini, Gazz. Chim. Ital., 36, 334 (1906); (c) G. Bargellini, Chem. Zentr., %, 326 (1906); (d) C. A. Bishoff, Ber., 33, 933 (1900).

(52) A. A. Aroyan and V. V. Darbinyan, Izv. Akad. Nauk Arm. USSR, Khim. Nauk, 16, 59 (1963).

(33) (a) M. Julia and G. Tschernoff, Bull. Soc. Chim. France, 934 (1957); (b) L. Haskelburg, J. Org. Chem., 12, 426 (1947); (c) M. Matell, Arkiv Kemi, 2» 437 (1954); (d) D. Osborne, R. L. Wain and R. Walker, J. Hort Sci., 2%, 44 (1952).

(54) M. S. Smith, R. L. Wain and F. Wightman, Ann. Appl. Biol., 39, 295 (1952).

(55) M. Melandri, A. Buttini and P. Galimberti, Boll. Chim. Farm., 102. 777 (1963).

(56) J. Koo, S. Avakian and G. J. Martin, J. Am. Chem. Soc., 77, 5373 (1955). 6h (57) R. Kurdukar and N.V.S. Rao, Proc. Indian Acad. Sci., Sect. A,, 58, 336 (1963).

(58) J. S. McIntyre, Can. J. Chem., 767 (1967).

(59) L. F. Fieser and M. Fieser, J. Am, Chem. Soc., 54, 4347 (1932).

(60) D. C. Schroeder, P. 0. Corcoran, C. A. Holden and M. C. Mulligan, J. Org. Chem., 27, 586 (1962).

(61) F. Sondheimer and D. Rosenthal, J. Am. Chem. Soc., M , 3995 (1958).

(62) F. Pelizzou and G. Jommi, Ann. Chim. (Rome), 1461 (1959).

(63) M. S. Newman and P. E. Beal, III, J. Am. Chem. Soc., 72, 5163 (1950).

(64) D. T. Witiak, T. C.-L. Ho, W. E. Connor and R. E. Hackney, J. Med. Chem., 11, 1086 (1968).

(65) D. T. Witiak and M. W. Whitehouse, unpublished results.

(66) D. T, Witiak and M. W. Whitehouse, Biochem. Pharmacol., IE, 971 (1969).

(67) D. T. Witiak, T. D. Sokoloski, M. W. Whitehouse and F. Hermann, J. Med. Chem., 12, 754 (1969).

(68) D. T. Witiak, R. E. Hackney and M. W. Whitehouse, J. Med. Chem., 12, 697 (1969).

(69) D. H. Huffman,and D. L. Azarnoff, Steroids, £, 41 (1967).

(70) M. L. Black, G. Rodney and D. B. Capps, Biochem. Pharmacol., 17, 1803 (1968).

(71) L. L. Abell, B. B. Lavy, B. B. Brodie and F. E. Kendell, J. Biol. Chem., 195, 357 (1952).

(72) N.L.R. Bucher, J. Am. Chem. Soc., 75., 498 (1953).

(73) A. Fredge, Acta Chem. Scand., 9, 719 (1955).

(74) H. E. Zaugg, D. A. Dunnigan, R. J. Michaels, L. R. Swett, T. S. Wang, A. H. Sommers and R. W. DeNet, J. Org. Chem., 26, 644 (1961).

(75) H. Conroy, J. Am. Chem. Soc., 74, 3046 (1952).

(76) F. Sondheimer and D. Rosenthal, J. Am. Chem. Soc., M , 3995 (1958). 65 (77) L. Caglioti and P. Grasselli, Chem. Ind., 153 (1964).

(78) L. A. Brooks and H. R. Snyder, Org. Syn., Coll. Vol., 3, 698 (1955).

(79) D. Misti, F. D. Marchi and V. Rosnati, Gazz. Chim. Ital., 93, 52 (1963).

(80) W. L. Holmes and J. D. Bentz, J. Biol. Chem., 235, 3118 (1960).

(81) R. G. Langdon and K. Bloch, J. Biol. Chem., 200, 129 (1953).

(82) B. Sjoberg, Arkiv Kemi, 15^, 451 (1960).

(83) F. A. Bovey, in "Nuclear Magnetic Resonance Spectroscopy," Academic Press, Inc., New York, N. Y., 1969, pp. 105.