The Synthesis and Preliminary Pharmacological Screening of Some Benzoyl Diamides of Ethylenediamine and Piperazine

AN ABSTRACT OF THE THESIS OF Pharmaceutical Richard Eugene Lindgren, Jr. for the M.S. in Chemistry (Name) (Degree) (Major)

Date thesis is presented 7/,,,,,e-c . l6 /963

Title THE SYNTHESIS AND PRELIMINARY PHARMACOLOGICAL

SCREENING OF SOME BENZOYL DIAMIDES OF

ETHYLENEDIAMINE AND PIPERAZINE

Abstract approved_ Redacted for Privacy (Major Professor)

Twelve N, N' -bis(hydroxy- . and methoxybenzoyl)ethylenedi - amines and piperazines have been prepared, tested for antifungal activity, and screened for gross pharmacological activity in mice.

All twelve compounds showed some activity against Candida albicans,

Trichophyton mentagrophytes and Microsporum gypseum but in ev- ery case the activity was less than that of salicylanilide used as a control. In the test on mice N, N' -bis(4-hydroxybenzoyl)ethylene- daimine and N, N' -bis(3- methoxybenzoyl)piperazine showed activity (stimulation) after oral administration. THE SYNTHESIS AND PRELIMINARY PHARMACOLOGICAL SCREENING OF SOME BENZOYL DIAMIDES OF ETHYLENEDIAMINE AND PIPERAZINE

RICHARD EUGENE LINDGREN, JR.

A THESIS

submitted to

OREGON STATE UNIVERSITY

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

June 1966 APPROVED Redacted for Privacy Assistant Professor of PharVnaceuticál Chemistry

In Charge of Major

Redacted for Privacy Head of Department of Pharmaceutical Chemistry

Redacted for Privacy

Dean of Graduate School

Date thesis is presented t'- t'/,y /¡y /l /%,-S Typed by Gail Dailey TABLE OF CONTENTS Page

INTRODUCTION 1

METHODS OF PREPARATION 11

EXPERIMENTAL 14

Synthesis 14 Antifungal Testing In Vitro 24 Results of Antifungal Activity 25 Gross Activity In Vivo 28 Results of In Vivo Study 29

SUMMARY 30

BIBLIOGRAPHY 31 LIST OF TABLES

Table Page

I. Antifungal Activity of Diamides of Ethylenediamine 26

II. Antifungal Activity of Diamides of Piperazine 27

III. Antifungal Activity of Controls 28 THE SYNTHESIS AND PRELIMINARY PHARMACOLOGICAL SCREENING OF SOME BENZOYL DIAMIDES OF ETHYLENEDIEMINE AND PIPERAZINE

INTRODUCTION

The amide linkage is found in a number of pharmacological agents of wide therapeutic activity and varying molecular complexity.

Among the activities for which amides have been studied are sedative - hypnotic, spasmolytic, fungistatic, analgesic, antipyretic, and local anesthetic. It would be reasonable to expect that diamides might also have similar activities.

The following discussion concerns monoamides. In most in- stances the limits of molecular variation for each pharmacological activity in the series have been outlined as well as the determination of the compound in the series with the optimum activity. Molecular variation as related to each individual pharmacological activity of amides is discussed below.

The amides possessing sedative -hypnotic activity generally can be considered as derivatives of acetamide. Increased sedative -hypnotic effect has been obtained by replacing two (4, 29) or three (4, 14,

29) of the hydrogens on the a- carbon acetamide with short chain and/ or unsaturated groups (14). Substitution of two alkyl groups and a chlorine or bromine for the hydrogens of the a- carbon also increased activity (4, 29). A cyclic group connected to acetamide through a 2

single carbon linkage and by substitution in the ortho position of the cyclic substituent also enhanced sedative -hypnotic activity and was decreased by substitution in the meta or para position (22).

Chapman et al (1 1) reported phenyldiethylacetamide to be about twice as effective in producing narcosis but with greater toxicity than

3- methyl -l- pentyn -3 -ol. Chloro and methyl substitution on the phenyl group in the ortho position increased both narcosis and acute toxicity as compared to substitution on the meta and para isomers. Para chlorination of phenyldimethylacetamide increased narcosis but para chlorination of phenyldiethylacetamide decreased narcosis (11). Mar- tin, Brendel, and Beiler (20) found 2- allyl-2- benzylacetamide and 2- allyl-2-(l-phenylethyl) acetamide the most active of a series of sub-

stituted acetamides. Substitution of methallyl or amyl decreased activity as did substitution of a methyl or methoxy group on the 1- phenylethyl group. They also noted that some unsaturated substituted acetamides, although inactive alone, were effective when used with pento- barbital.

Junkmann in a study of the spasmolytic effects of substituted acetamides found that alkyl amides ranging from twelve to seventeen carbon atoms gave optimum activity (14). In a further study (15) he found five of these compounds more active spasmolytics against bar- ium chloride induced spasm on rabbit intestine than papaverine. They were, in decreasing order of activity: diamylethylacetarnide, 3 triamylacetamide, dihexylmethylacetamide, tributylacetamide, and dibutylacetamide. Evidence of skeletal muscle relaxing effects have been noted in salicylamide and its derivatives (1, 21). Matsumura

(21) reported N- methyl and N, N- dimethylsalicylamide and N, N -dimethyl 2- ethoxybenzamide had less muscle relaxing effect than salicylamide. He suggested that the presence of a hydroxy or alkoxy group in the ortho position of a nonsubstituted acid amide group was required for activity.

A high order of fungistatic activity has been reported for bromoacetamides with N- phenyl or N -alkyl substituents containing four to ten carbon atoms (18). Bromine as a substituent was found more active than chlorine. Primary N- alkyls were found more active than branched chain N- alkyls and N- monosubstituted compounds were found more active than disubstituted compounds.

Investigation of the fungicidal activity of amides has centered largely about salicylamides and the long chain fatty acid amides. The fungicidal activity of fatty acid amides is enhanced by increasing chain length or unsaturation but the fungicidal activity is less as a group than the corresponding acids (37). Salicylanilide has been reported as effective as undecylenic acid in the treatment of tinea captis caused by Mycrosporum audonini(30) Coates et al (12) found 2 -n- amyloxybenzamide the most active of a series of alkyl ethers of salicylamide. Alkyl ethers of three to seven carbon range were found to 4 be fungistatic with 2- n- hexyloxybenzamide being more active when the incubation period was extended to fourteen days. N- Substitution did not seem to enhance activity nor was activity increased by nuclear halogen substitution. Also the naphthamide analogs were not as active.

Analgesic activities have been found in some aromatic amides such as N- phenylacetamide (acetanilid), N- acetyl- 4- ethoxyaniline

(phenacetin) and salicylamide. According to Brodie and Axelrod (5) acetanilid is metabolized to N- acetyl -4- aminophenol which has been suggested as being the active form of the analgesic. Although p- aminophenol possesses high analgesic activity it is not used as a drug because of its high toxicity.

The analgesic activity of salicylamide has been found to be generally reduced by introduction of a methyl or acetyl group on the aromatic hydroxyl group (8). However substitution of the hydroxyl group of salicylamide by ethoxy (16, 32, 36) or allyl (32, 36) doubles or tri- ples the analgesic activity of salicylamide. Substitution on the benzene ring of salicylamide in the three -, four -, or five- positions by phenyl, bromo, hydroxyl, or methoxyl groups generally decreased analgesic potency and toxicity (36). A notable exception was 3- phenyl salicylamide which was found to be a less toxic and more potent analgesic than salicylamide (36). Substitution on the nitrogen portion of the salicylamide molecule by (3 -hydroxyethyl or dimethyl groups increased potency as well as toxicity (36). Way et al (36) found 5 toxicity but not always potency was increased by N- methylation, N- ethylation, or N, N- diethylation. Carron, Tabert, and Jullien (8) found that alkyls from ethyl to isopropyl increased analgesic activity but that N- methyl and N -butyl derivatives lowered activity. Tamori,

Nishio, and Kumura (32) found that N- cyclohexylation reduced the activity of salicylamide derivatives,

In a study of the antiypretic action of salicylate and gentisate derivatives, Preziosi (27) found N, N- diethylsalicylamide and 2- ethoxybenzamide to be more effective than salicylamide when tested intraperitoneally upon pyrexic rabbits. Kurbjuweit, Kroneberg, and

Sprangler (16) also found the antipyretic action of 2- ethoxybenzamide greater than that of salicylamide. However, Carlsson and Magnusson

(7) found that pyrexic rabbits showing antipyretic response to aspirin gave no detectable antipyretic response to salicylamide, 2- ethoxybenzamide, or 2- propoxybenzamide.

The local anesthetic activity of amides has received consider- able attention. In a study of local anesthetic activity, Villiani, Lane, and Papa (35) found that N' -benzyl -N' - cinnamoyl -N, N- diethyl ethyl - enediamine had the most prolonged activity of the series. Para substitution on the aromatic ring with nitro, amino, chloro, or isopropyl or substitution on the a- carbon of cinnamic acid decreased activity by one -third to two -thirds. Replacement of diethyl by dimethyl reduced the duration of action. Kurihara et al (17) investigated a series of 6 nuclear halogenated, alkoxylated benzamides as local anesthetics.

They found the amide derivatives of 2- alkoxy -4- bromobenzoic acid and 3- bromo -4- alkoxybenzoic acid to exhibit the greatest activity. In these cases the alkoxy group was ethoxy to butoxy and the dialkylamino group was dipropylamino, dibutylamino, or piperidine. Dibucaine, which was introduced by Uhlman in 1929 (33), and lidocaine, patented in this country by Lofgren and Lundqvist in 1948 (19), are examples of commercially available local anesthetics containing the amide linkage.

In addition to the above activities, the amide linkage appears in compounds having various other pharmacological activities. Included are such well known antibiotics as bacitracin, chloramphenicol, gra- micidin, penicillin and its derivatives, and tetracycline and its derivatives. Antiemetic activity has been also reported; for example, 4-

(2- dimethylaminoethoxy) -N -(3, 4, 5 -trimethoxybenzoyl)benzylamine,

1 trimethoxybenzamide .

In contrast to the extensive pharmacological investigation of amides, the diamides have received little attention. On the basis of previously shown activities of amides it would appear that diamides might also possess similar pharmacological activities. The present survey is limited to symmetrical diamides formed from diamino

1Marketed as Tigan® by Roche Laboratories, Nutley, N. J. 7 compounds. Although these compounds have been investigated for nu- merous activities such as: antiemebic, spasmolytic, sedative, analgesic, antipyretic, estrogenic, antitubercular, and anthelmintic activities, the literature surveyed revealed no extensive study in this area.

Berberian, Slighter, and Surrey (3) prepared a series of N, N' - bis(dichloroacetyl)diamides for antiemebic activity. In these compounds the interamide chain varied from C1 to C10. N, N' -Dihalo- acetyl-N, N' -dialkylamines were found more active in both in vitro and in vivo tests than the corresponding secondary amides.

Pimenta et al (24) investigated N, N' -bis(3, 4, 5- trimethoxybenzoyl)ethylenediamine as one of a series of four compounds to deter- mine the effect of substitution of the amide linkage for the ester linkage in compounds of the reserpine type. All compounds showed spas - molytic action on smooth muscle, transitory hypotensive action in the dog, and cardiac arrest in isolated frog heart.

N, N' -Bis (dialkylacetyl)ethylenediamines showed no practical sedative or analgesic activity in tests on rabbit tooth pulp (6). These compounds were of interest because they contain the -C -CO -NH group of the barbiturate and acetamide series. Carron, Tabert, and Jullien

(8) reported two salicylamide molecules joined by a three carbon chain between the nitrogens increased the analgesic effect of salicylamide but that a one or two carbon chain lowered analgesic activity. It has been reported that N, N' -bis(2- hydroxybenzoyl )hexamethylenediamine 8 and N, N' -bis( 2 -hydroxybenzoyl)ethylenediamine have both analgesic and antipyretic activity (28).

Miguel, Barany, and Muller (23) screened a series of compounds including N, N' -bis( 4- hydroxybenzoyl)ethylenediamine for hor- mone antagonism but found this compound to have little estrogenic activity.

Pollard and Adelson (25) prepared a series of diamides of piper - azine, including the phenacetyl, hydrocinnamoyl, and anisoyl derivatives for general physiological testing. However the results of the physiological testing were not reported.

Since N, N' -bis(benzenesulfonyl)piperazine (31) had marked activity in inhibiting the growth of tuberculi bacillus, Pollard and Gray

(26) prepared a series of twelve diamides of piperazine including N,

N' -bis(2- and 4- chlorobenzoyl)piperazine but found all the diamides tested to be ineffective against tuberculi bacillus.

A number of N, N'- diacylpiperazine derivatives have been prepared as potential anthelmintic agents (9, 13). N, N' - Dicaproyl -N,

N' -dienanthyl- and N, N' -bis(monochloroacetyl)piperazine were reported as being of therapeutic interest (13). Dibenzoyylpiperazine was reported to have little anthelmintic activity (9, 13) and was found to be relatively toxic in mice (9).

Cerbai, Di Paco, and Dell'Omodarme (10) reported the preparation of N, N' -bis(3, 4, 5 -trimethoxybenzoyl)piperazine as a part of a 9 study of the hypnotic and hypotensive effects of a series of heterocyclic compounds.

As the hydroxyl and alkoxyl derivatives of benzamide have shown a wide range of physiological activity it might be expected that diamides of these compounds might also show activity. The hydroxylated and alkoxylated benzoyl diamides of ethylenediamine and piperazine contain not only the substituted benzamide moiety but also the ethylenediamine and piperazine basic moiety which is present in a number of pharmacologically active compounds. Compounds containing the ethylenediamine moiety include the antihistamines tripelen- amine2 and pyrilamine3 as well as the antiarrythmic drug procain- amide hydrochloride4 and the local anesthetic dibucaine hydrochloride. 5 Compounds containing the piperazine moiety include the anti-

7 histamine chlorcyclizine hydrochloride, 6 the antinauseant meclizine, and the ataraxic drug hydroxyzine hydrochloride. 8 It was the purpose

2Marketed as Pyribenzamine® by Ciba Pharmaceutical Co. , Summit, N. J. 3Marketed as Neo- Antergan® by Merck Sharp and Dohme, West Point, Pennsylvania. 4Marketed as Pronestyl® by E. R. Squibb, New York, N. Y.

5Marketed as Nupercaine® Hydrochloride by Ciba Pharmaceutical Co. , Summit, N. J. 6 Marketed as Perazil® Hydrochloride by Burroughs Wellcome and Co. , Tuckahoe, N. Y. 7Marketed as Bonine® by Pfizer Laboratories, New York, N. Y.

8Marketed as Atarax® Hydrochloride by J. B. Roerig and Company, New York, N. Y. 10 of this study to synthesize and to preliminarily screen compounds containing both the hydroxylated and alkoxylated benzamide moiety and the ethylenediamine or piperazine moiety for possible pharmacological activity. 11

METHODS OF PREPARATION

Of the well known methods for preparation of amides, the general method followed here was the reaction of an aroyl chloride with the appropriate amine as shown in the following reactions:

O O, O C-OH SO C12 C-C1 acetyl chloride O O OH 02C-CH 0-6-CH3 3

O -ci O H HQ ethylenediamine óNCH2CH2NC. O O o O-C-CH3 O-C-CH3 /I O-C -CH3

9 H H A 9 HO CNCH2CH2NC N4OH CNCH2CH2NC í 0 > OC-CH3 O-C-CH3 t.-OH OH \% 4

Compounds prepared in this manner were

N, N' -bis(3- hydroxybenzoyl)ethylenediamine and

N, N' -bis(4- hydroxybenzoyl)ethylenediamine.

Analogous steps in which the amine was piperazine were used in the preparation of

N, N'- bis(3- hydroxybenzoyl)piperazine and

N, N' -bis(4- hydroxybenzoyl)piperazine. 12

In the preparation of

N, N' -bis(2- hydroxybenzoyl )pipe razine the readily available aspirin eliminated the need for the first step of

synthesis. In the preparation of

N, N' -bis (2- hydroxybenzoyl)ethylenediamine the readily available methyl salicylate was refluxed with ethylenediamine for eight hours to give the product.

O H II OH p C-O-CH3 CNCH CH NC í -OH ethylenediamine OH2 2 - OH I v

The methoxy derivatives were prepared by the same general

procedure as shown in the following reactions:

O O C-OH O C-Cl C-OH \ (CH 3) 2SO4 SO C12 / \\ OH OCH3 OCH ' 3 ,_%z O OH HO C-C1 ethylenediamine `NCH2CH2NC OCH OCH3 -OCH3 3

Compounds prepared in this manner were

N, N' -bis( 2- methoxybenzoyl)ethylenediamine and

N, N' -bis( 3- methoxybenzoyl)ethylenediamine. 13

Analogous steps in which the amine was piperazine were used in the preparation of

N, N' -bis(2- methoxybenzoyl)piperazine and

N, N' -bis (3- methoxybenzoyl)piperazine.

In the preparation of

N,N' -bis(4- methoxybenzoyl)ethylenediamine and

N, N' -bis(4- methoxybenzoyl)piperazine the readily available anisic acid eliminated the need for the first step of synthesis. 14

EXPERIMENTAL1' 2

Synthesis

N, N' -Bis( 2- hydroxybenzoyl)ethylenediamine

A solution of three g. (0.05 mole) of ethylenediamine and 15.2 g. (0. 1 mole) methyl salicylate was refluxed eight hours. The crude product was dissolved in ethanol and precipitated by the addition of water. The solid material was recrystallized from ethanol. The yield was 5.5 g. (36 %) of white crystalline material, melting range

177-178°. Van Allen (34) reports a melting range of 183-184°.

Anal. Calc'd. for C16 H16 N2 04:

C 63.99 H 5.38 N 9.33 Found:

C 64.12 H 5.27 N 9.21

1 All melting points were obtained by the use of a Thomas Hoover melting point apparatus.

2Carbon, hydrogen, and nitrogen determinations were conducted by Dr. G. Weiler and Dr. F. B. Strauss, Microanalytical Labor- atory, 164, Banbury Road, Oxford, England. 15

N, N' -Bis(2-hydroxybenzoyl)piperazine

A mixture of 23 g. (0.13 mole) of aspirin, 45 ml. of benzene and 54 ml. thionyl chloride was refluxed for one hour. The excess thionyl chloride and benzene was removed by reduced pressure. The fraction distilling from 148 -151025 mm. weighed 17 g. (72 %). This fraction was dissolved in 38 ml. benzene and cooled on an ice bath.

Piperazine (3. 9 g. , 0.05 mole) in pyridine was added dropwise with stirring. The resultant mixture was refluxed on a steam bath for one hour and then placed in an evaporating dish and allowed to evaporate.

The remaining syrupy material was dissolved in 100 ml. of ten percent sodium hydroxide solution and acidified with concentrated hydrochloric acid. The precipitate was washed with water and allowed to dry, yielding 6. 7 g. (45 %) of a white powder, melting range 313 -314°.

Anal. Calc'd. for C18 H18 N2 04:

C 66.24 H 5.57 N 8.58 Found:

C 65.88 H 5.53 N 8.51

3- Acetoxybenzoic acid

A mixture of five g. (0.036 mole) 3- hydroxybenzoic acid, 17 ml. acetic anhydride and two ml. pyridine was shaken and allowed to stand for thirty minutes. The mixture was poured into 80 ml. of ice water and stirred. The acetoxylated acid formed as a white 16 precipitate having a melting range of 128-132°. Anschutz and Mot - schmann (2) reported a melting point of 131.5°.

N, N'- Bis( 3- hydroxybenzoyl)ethylenediami.ne

A mixture of five g. (0.03 mole) 3- acetoxybenzoic acid, 20 ml. thionyl chloride and 20 ml. benzene was refluxed two and one -half hours. The excess thionyl chloride was removed in vacuo at 1000.

Two 20 ml. portions of toluene were added successively and distilled in vacuo at 1000. The solid residue was dissolved in benzene and cooled on an ice bath. Ethylenediamine (one g., 0. 017 mole) in pyridine was added dropwise with shaking, then the mixture was placed in an evaporating dish and allowed to evaporate to dryness. The solid residue was allowed to stand in ten percent sodium hydroxide solution for three hours. Acidification of the resulting solution with concentrated hydrochloric acid yielded 1.2 g. (20%) of a white powder, melting range 245-246°,

Anal. for C16 Calc'd. H16 N2 04:

C 63.99 H 5. 38 N 9. 33 Found:

C 62.53 H 5. 23 N 9. 63 17

N, N' -Bis(3-hydroxybenzoyl)piperazine

A mixture of five g. (0. 03 mole) 3- acetoxybenzoic acid, 20 ml. thionyl chloride and 20 ml. benzene was refluxed two and one -half hours. The excess thionyl chloride was removed in vacuo at 1000.

Two 20 ml. portions of toluene were added successively and removed in vacuo at 100o. The crude product was dissolved in benzene and cooled in an ice bath. A ten percent solution of pyridine in chloroform was prepared and 1.34 g. (0. 016 mole) of piperazine was dissolved in it. This solution was added dropwise with stirring to the above benzene solution. The mixture was placed in an evaporating dish and allowed to evaporate to dryness. The resulting solid residue which formed was dissolved in ten percent sodium hydroxide solution.

After standing three hours it was acidified with concentrated hydrochloric acid, yielding 1.9 g. (26 %) of a buff powder, melting range

309 -3110 (decomposition).

Anal. Calc'd. for C18 H18 N2 04:

C 66.24 H 5.57 N 8.58 Found:

C 66.26 H 5.82 N 8.62 18

N, N' -Bis (4 -hydroxybenzoyl )ethylenediamine

4- Hydroxybenzoic acid weighing 18 g. (0. 13 mole) was acetyl - ated with acetyl chloride, yielding 18 g. (74 %) of crude product. A mixture of the crude 4- acetoxybenzoic acid was dissolved in an excess of thionyl chloride and benzene and the mixture was refluxed one hour.

The excess thionyl chloride and benzene was removed in vacuo at 0 100 to give ten g. (0. 05 mole) of the crude acid chloride. This material was dissolved in benzene and reacted (by slow addition) with

1.5 g. (0. 025 mole) ethylenediamine in pyridine, and the resulting mixture was then placed in an evaporating dish and allowed to evaporate to dryness. The resulting solid residue which formed was dissolved in ten percent sodium hydroxide solution. After standing three hours it was precipitated by acidification with concentrated hydrochloric acid. The precipitate was recrystallized from methanol - water. There was obtained 6. 7 g. (88 %) of a white powder, melting range 236-238°. It was noted that Miguel, Barany, and Muller (23) reported a melting point of 190°.

Anal. Calc'd. for C16 H16 N2 04:

C 63.99 H 5.38 N 9.33 Found:

C 64.11 H 5.44 N 9.13 19

N, N' -Bis(4-hydroxybenzoyl)piperazine

Fourteen g. (0.1 mole) of 4- hydroxybenzoic acid was acetylated with acetyl chloride, yielding 13.5 g. (71 %) of crude product. A mixture of the crude 4- acetoxybenzoic acid was dissolved in benzene and an excess of thionyl chloride, and the mixture refluxed one hour. The excess thionyl chloride and benzene was removed in vacuo at 100o.

The crude product was dissolved in 25 ml. chloroform and reacted

(by slow addition) with three g. (O. 035 mole) of piperazine in 120 ml. of five percent sodium hydroxide. The solid material was separated by filtration. The crude ester was hydrolyzed by warming in 100 ml. of ten percent sodium hydroxide solution until the material dissolved.

The solution was filtered and acidified with concentrated hydrochloric acid. An excess of sodium bicarbonate was added and the solid material washed with water and dried. There was obtained 6. 7 g. (58 %) of white powder, melting range 321-322°.

Anal. Calc'd. for C18 H18 N2 04:

C 66.24 H 5.57 N 8.58 Found:

C 65.50 H 5.53 N 8.65 20

N, N' -Bis( 2- methoxybenzoyl)ethylenediamine

A mixture of 23 g. (0.15 mole) 2- methoxybenzoic acid, benzene,

and 67 ml. thionyl chloride was refluxed one and one -half hours. The

excess thionyl chloride and benzene was removed in vacuo at 1000.

The crude product was dissolved in 25 ml. of benzene and slowly

added with stirring to a solution of one g. (0. 017 mole) ethylenedi-

amine in eight ml. of pyridine. The resultant mixture was placed in

an evaporating dish and allowed to evaporate to dryness. The solid

residue was powdered finely and washed with ethanol. The residual material was recrystallized from toluene. There was obtained 5.0

g. (99 %) of a white powder, melting range 137-138°.

Anal. Calc'd. for C18 H2O N2 04:

C 65.83 H 6.15 N 8.53 Found:

C 66.75 H 6.24 N 8.18

N, N' -Bis(2-methoxybenzoyl)piperazine

A mixture of 23 g. (0. 15 mole) 2- methoxybenzoic acid, benzene,

and 50 ml. thionyl chloride was refluxed for one hour. The excess thionyl chloride and benzene was removed in vacuo at 1000. The

crude product was dissolved in benzene and slowly added to six g. (0.07 mole) piperazine in pyridine. The resulting mixture was 21 placed in an evaporating dish and allowed to evaporate to dryness.

The solid residue was recrystallized from dimethylformamide by the addition of water. The yield was 1.4 g. (6 %) of a white powder, melting range 292 -294 °.

Anal. Calc'd. for C20 H22 N2 04:

C 67.77 H 6.27 N 7.90 Found:

C 66.05 H 5.83 N 8.24

N, N' -Bis( 3 -methoxybenzoyl)ethylenediamine

A mixture of five g. (0. 033 mole) 3- methoxybenzoic acid and

22 ml. thionyl chloride was refluxed for one hour. The excess thionyl chloride was removed in vacuo at 100°. Toluene in two successive 25 ml. portions was added and removed in vacuo at 100 °.

The crude product was dissolved in benzene and one ml. (0. 017 mole) of ethylenediamine in pyridine was slowly added with shaking and cooling. Recrystallization from ethanol yielded 1.5 g. (27 %) of white crystals, melting range 165 - 166 °.

Anal. Calc'd. for C18 H2O N2 04:

C 65.83 H 6.15 N 8.53 Found:

C 65.63 H 6.43 N 8.45 22

N, N' -Bis(3-methoxybenzoyl)piperazine

A mixture of 2.5 g. (0. 016 mole) 3- methoxybenzoic acid and 13 ml. thionyl chloride was refluxed two hours. The excess thionyl chloride was removed in vacuo at 100e. Toluene in two successive portions was added and removed in vacuo at 1000. The solid residue was dissolved in benzene and slowly added to 0. 69 g. (0. 008 mole) piperazine in pyridine. The resultant mixture was placed in an evaporating dish and allowed to evaporate. Recrystallization from isopro- panol yielded 1.5 g. (53 %) of a white powder, melting range 161.5-

1630.

Anal. Calc'd. for C20 H22 N2 04:

C 67.77 H 6.27 N 7.90 Found:

C 68.05 H 6.45 N 8.13

N, N' -Bi s (4 -methoxybenzoyl)ethylenediamine

A mixture of 20 g. (0. 13 mole) anisic acid, benzene, and 54 ml. thionyl chloride was refluxed for one and one -half hours. The mixture was distilled in vacuo. The fraction distilling in the range 174 -

178042 mm. was collected, yielding 20 g. (0. 12 mole, 93 %) of anisoyl chloride. To ten g. (0. 06 mole) of anisoyl chloride in benzene was slowly added 1.8 g. (0.03 mole) ethylenediamine. The resultant 23 mixture was placed in an evaporating dish and allowed to evaporate.

The solid residue was washed once with water, twice with five percent sodium carbonate solution, and finally with water. The solid residue was recrystallized from dimethylformamide. There was obtained seven g. (73 %) of white crystalline material, melting range 221-223°.

Anal. Calc'd. for C18 H2O N2 04:

C 65.83 H 6.15 N 8.53

Found:

C 66.04 H 6.22 N 8.45

N, N' -Bis(4-methoxybenzoyl)piperazine

To ten g. (0.06 mole) anisoyl chloride in benzene 2. 6 g. (0.03 mole) of piperazine in pyridine was added dropwise with shaking. The resultant mixture was placed in an evaporating dish and allowed to evaporate. The solid residue was recrystallized twice from dimethylformamide. There was obtained 7.5 g. (78 %) of white crystalline material, melting range 189.5-191.5°. Pollard and Adelson (25) reported a melting range of 192.5-193.5°.

Anal. Calc'd. for C20 H22 N2 04:

C 67.77 H 6. 27 N 7.90

Found:

C 67.46 H 6.04 N 7.95 24

Antifungal Testing In Vitro

One percent ethanol solutions of the compounds to be tested were prepared. Those compounds soluble in one percent concentrations were also prepared as two -tenths percent solutions in ethanol. Those compounds not soluble in one percent concentration in ethanol were tested for activity as the one percent suspension and as the saturated ethanol solutions.

Petri dishes containing ten ml. of Sabouraud dextrose agar were sterilized in an autoclave at fifteen pounds pressure for thirty minutes.

After cooling, each plate was innoculated with one ml. of aqueous suspension of Candida albicans, Trichophyton mentagrophytes, or Micro - sporum gypseum. The Petri dishes were allowed to stand for one hour and three sterile filter discs were placed on each plate with a sterile forceps.

Two drops of a solution or a suspension of each of the chemi- cals to be tested were placed on each of the three discs on a given plate. Candida albicans and Trichophyton mentagrophytes cultures did not become well developed on the plates until the third day. Mea- surements of zones of inhibition were taken after three days. Sali- cylanilide and salicylic acid, common antifungal standards, were used as controls. 25

Results of Antifungal Activity

From the limited data shown in Table I and II it may be seen that all compounds showed some activity against Candida albicans, Trichophyton mentagrophytes, and Microsporum gypseum but less than that of the controls as shown in Table III.

The N, N' -bis(methoxybenzoyl)piperazines were the most active of the experimental compounds. However, as will be noted, the areas of inhibition are very narrow and the results should be considered qualitative rather than quantitative. No closely related compounds were found in the literature surveyed so that antifungal activity of related compounds could not be compared. 26

Table I

Antifungal Activity of Diamides of Ethylenediamine

O H H p ( C:-N-CH2-CHL-1\T__C - )/ \ _ /` R

Minimum widtha of zone of inhibition (in mm. ) Candida Trichophyton Microsporum R Concentration albicans mentagrophytes gypseum (w /v)

2-OH 1%b 1 0 0

2-OH 0.2%b 2 3 3

3-OH 1%c 0 1 0

3-OH d 2 0 2

4-OH 1%b 3 1 0

4-OH 0.2%b 2 3 0

2-OMe 1%b 2 2 2 2-OMe 0.2%b 2 3 2

3-OMe 1%c 2 2 2 3-OMe d 2 0 0

4-OMe 1%c 1 2 0

4-OMe d 2 3 1 a Average of three measurements b Ethanol solution c Ethanol suspension d Saturated ethanol solution 27

Table II

Antifungal Activity of Diamides of Piperazine

C -*N N-C

Minimum widtha of zone of inhibition (in mm. ) F Candida Trichphyton Microsporum R Concentration albicans mentagrophytes gypseum (w/v)

2-OH 1%c 1 0 0 2-OH d 4 2 2

3-OH 1%c 0 2 0

3-OH d 2 2 1

4-OH 1%c 1 3 0 .; 4-OH d 0 0 2

2-OMe 1%c 4 2 1

2-OMe d 2 3 1

3-OMe 1%c 3 3 0

3-OMe d 3 2 2

4-OMe 1%c 1 3 2 4-OMe d 3 2 0 a Average of three measurements c Ethanol suspension d Saturated ethanol solution 28

Table III

Antifungal Activity of Controls

Minimum widtha of zone of inhibition (in mm. ) Compound Concentration Candida Trichophyton Microsporum (w/v) albicans mentagrophytes gypseum

Ethanol 95% 0 0 o

Salicylic 1 %b 4 3 2 acid b Salicylic 0. 2 3 1 2 acid

Salicyl- 1 no growth growth on slight if anilide on plate outer rim any growth b Salicyl- 0.2% no growth growth on slight if anilide on plate outer rim any growth a Average of three measurements b Ethanol solution

Gross Activity In Vivo

A four percent suspension was prepared by suspending 100 mg. of each compound in 2.5 ml. of 0. 2% methylcellulose in water. By serial dilution 0.4 percent and 0. 04 percent suspensions were prepared. To facilitate administration the four percent suspension was diluted to a two percent suspension with the methylcellulose preparation in water and administered in one ml. volume. The 0.4 percent and the 0.04 percent suspensions were administered in 0.5 ml. 29 volume. Three mice were administered each concentration of one of the compounds. The dosage based on a mouse of twenty grams body weight would result in the following doses:

Two percent 1000 mgm. per kilogram suspension

0.4 percent 100 mgm. per kilogram suspension

0. 04 percent 10 mgm. per kilogram suspension

Results of in vivo study

Those mice administered the three suspensions of N, N' -bis(4- hydroxybenzoyl)ethylenediamine at all three dosage levels exhibited hyperactivity within fifteen minutes after administration and hyperactivity continued for two hours. Those mice administered suspensions of N, N' -bis(3- methoxybenzoyl)piperazine showed some increased hyperactivity. All other experimental compounds were inactive in the test. The mice were observed for three hours. 30

SUMMARY

Twelve N, N' -bis(hydroxy- and methoxybenzoyl)ethylenediamines and piperazines of which nine were new compounds have been prepared and tested for antifungal activity against Candida albicans, Tricho-

phyton mentagrophytes , and Microsporum gypseum. All twelve compounds showed some activity against these organisms but less than that of salicylanilide which was used as a control. Upon the administration to mice of oral suspensions at three dosage levels of the compounds only N, N' -bis(4- hydroxybenzoyl)ethylenediamine and N, N' - bis(3- methoxybenzoyl)piperazine induced hyperactivity. 31

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