SYNTHESIS OF SOME AIÆIDES OF

GENTISIO AOID

DISSERTATION

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

By

ALLEN I. DHŒS, B. S., M. Sc,

******

The Ohio State Uhiversity

1958

Approved by:

Department of Pharmaoy ACKNOV/LEDaMTS

I wish to acknowledge the generous advice and assistance rendered by Dr. Frank V/. Bope, Associate

Professor of Pharmacy, in the development of this dissertation.

To the American Foundation for Pharmaceutical

Education, I wish to express my gratitude for extending to me financial assistance in the form of a Fellowship— without this assistance it would not have been possible for me to complete my graduate studies.

To the members of the faculty of the College of

Pharmacy of The Ohio State University, I wish to express my sincere appreciation for their guidance throughout my graduate studies,

I also wish to eatress my sincere thanks and grate­ ful appreciation to all others who have assisted me in any way.

As a final note, I would like to thank my wife,

Charlotte, for her wonderful moral support during the com­ pletion of my graduate studies; for behind every man there is his wife.

ii TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

DISCUSSION OF LITERATURE

General ...... 3 Some New Gentisio Aoid Derivatives 15 Preparation of Amides ...... 16

GENERAL PROCEDURES 21

EXPERIMENTAL 23

Preparation of 2,5- Dimethoiybenzoio Acid . . , 23 Preparation of 2,5- Dimethoxybenzoyl Chloride 26 Classification of N- Substituted Amides Prepared 27 Monosubstituted Amides of 2,5- Dimethoxybenzoio A o i d ...... 29 Alkyl ...... 29 N- n- Propyl- 2,5- Dimethoxybenzamide . . . 29 N- n- Butyl- 2,5- Dimethoxybenzamide . . . . 31 N- Isoamyl- 2,5- Dimethoxybenzamide .... 32 Dialkylaminoalkyl...... 33 N - y - Dimethylaminopropyl- 2,5- Dimethoxy­ benzamide Hydrochloride ...... 33 N - y - Diethylaminopropyl- 2,5- Dimethoxy­ benzamide Hydrochloride ...... 34 Alicyclio ...... 36 N- Cyclohexyl- 2,5- Dimethoxybenzamide . , . 36 Aromatic ...... 37 N- 2*- Methylphenyl- 2,5- Dimethoxybenzamide 37 N- 5* - Methylphenyl- 2,5- Dimethoxybenzamide 39 N- 4»- Methylphenyl- 2,5- Dimethoxybenzamide 40 N- 2*,6 * - Dimethylphenyl- 2,5- Dimethoxy­ benzamide ...... » 41 N- 2»,5*- Dimethylphenyl- 2,5- Dimethoxy­ benzamide ...... 41 N- 2*,4'- Dimethylphenyl- 2,5- Dimethoxy­ benzamide ...... •...... 42 N- p- Diethylaminophenyl- à,5- Dimethoxy­ benzamide Hydrochloride ...... 43 Aralkyl ...... 44 N- Benzyl- 2,5- Dimethoxybenzamide...... 44 N- ^ - Phenylethyl- 2,5- Dimethoxybenzamide 46 iii Page

Heterooyolio A l k y l ...... 47 N - y - (4- Morphollno) propyl- 2,5- Dimethoxy­ benzamide Hydroohloride ...... 47 Disubstituted Amides of 2,5- Dimethoxybenzoio Aoid 49 D i a l k y l ...... 49 N,N- Diisobutyl- 2,5- Dimethoxybenzamide . . . 49 N,N- Diisoamyl- 2,5- Dimethoxybenzamide . . . 51 D i a r a l k y l ...... 52 N,N- Dibenzyl- 2,5- Dimethoxybenzamide .... 53 Misoellaneous Amides of 2,5- Dimethoxybenzoio Aoid 53 N- (2,5- Dimethoxybenzoy1) pyrrolidine .... 53 N- (2,5- Dimethoxybenzoyl) piperidine .... 55 2,5- DimethoxybenzoylUr e a ...... 56

INFRARED SPECTROSCOPY ...... 59

INFRARED SPECTROGRAPHS MD THEIR INTERPRETATION . . 65

OPTICAL PROPERTIES...... 100

DISTRIBUTION S T U D Y ...... 102

SUÎMARY AND DISCUSSION...... 109

CONCLUSIONS...... 122

LIST OF REFERENCES...... 125

AUTOBIOGRAPHY...... 130

±7 LIST OF TABLES

Table Page

I. Absorption Bands for Known Groups of Atoms as Compiled by The Ohio State University , . . 64

II. Absorption Bands Found in 8,5- Dimethozy- benzoio A c i d ...... 68

III. Absorption Bands Found in N- Alkyl Substi­ tuted Amides of 2,5- Dimethoxybenzoio Aoid 73

IV. Absorption Bands Found in N- Dielkylamino- alkyl Substituted Amides of 2,5- Dimethoxy benzoic Aoid ...... 76

V. Absorption Bands Found in N- Cyclohexyl- 2,5 Dimethoxybenzamide ...... 78

VI. Absorption Bands Found in N- Aromatic Substi tuted Amides of 2,5- Dimethoxybenzoio Acid 86

VII. Absorption Bands Found in N- Aralkyl Substi­ tuted Amides of 2,5- Dimethoxybenzoio Acid 90

VIII. Absorption Bands Found in N- - (4- Morpho- lino) propyl- 2,5- Dimethoxybenzamide . . 91

IX. Absorption Bands Found in N- Disubstituted Amides of 2,5- Dimethoxybenzoio Aoid . . 97

X. Absorption Bands Found in N- (2,5- Dimethoxy benzoyl) Piperidine and N- (2,5- Dimethoxy benzoyl) Pyrrolidine ...... 98

XI. Absorption Band Found in 2,5- Dimethoxyben­ zoyl urea ...... 98

XII. Alpha and Gamma Refractive Indices of the Crystalline N- Substituted Amides # # # 101

XIII. Distribution Coefficients (K) Obtained with N- y - Dimethylaminopropyl- 2,5- Dimethoxy benzamide .*...... • . • . 105 Table Page

TTT, Distribution Coefficients (K) ...... 107

3CV, Summary of Mono substituted Amides of 2,5- Dimethoxybenzoio Aoid P r e p a r e d ...... 117

z n . Summary of N- Disubstituted Amides of 2,5- Dimethoxybenzoio Aoid P r e p a r e d ...... 118

X7TI. Summary of Misoellaneous Amides of 2,5- Dimethozybenzoio Aoid Prepared ...... 118

vi LIST OF FIGURES

Figure Page

1. Infrared Spectrograph of 2,5- Dimethoxyhenzoic A o i d ...... 68

2. Infrared Spectrograph of N- n- Propyl- 2,5- Dimethoxybenzamide...... 70

3. Infrared Spectrograph of N- n- Butyl- 2,5- Dimethoxybenzamide . . . • ...... 71

4. Infrared Spectrograph of N- Isoamyl- 2,5- Dime thoxyb enzamide...... 72

5. Infrared Spectrograph of N- y - Dimethylamino­ propyl- 2,5- Dimethoxybenzamide Hydro­ chloride ...... 74

6 . Infrared Spectrograph of N- y - Diethylamino­ propyl- 2,5- Dimethoxybenzamide Hydro­ ohloride ...... 75

7. Infrared Spectrograph of N- Cyclohexyl- 2,5- Dimethoxybenzamide...... 77

8 . Infrared Spectrograph of N- 2»- Methylphenyl- 2.5- Dimethoxybenzamide ...... 79

9. Infrared Spectrograph of N- 3 Methyl phenyl- 8 .5- Dimethoxybenzamide ...... 80

10. Infrared Spectrograph of H- 4*- Methylphenyl- 2.5- Dimethoxybenzamide...... 81

11. Infrared Spectrograph of N- 2',6 '- Dimethyl­ phenyl- 2,5- Dimethoxybenzamide...... 82

12. Infrared Spectrograph of N- 2*,5*- Dimethyl­ phenyl- 2,5- Dimethoxybenzamide...... 83

13. Infrared Spectrograph of N- 2*,4*- Dimethyl­ phenyl- 2,5- Dimethoxybenzamide...... 84

Tii Figure Page

14. Infrared Speotrograpli of N-/»- Diethylamino­ phenyl- 2,5- Dimethoiyhenzamide Hydro­ ohloride ...... « ...... 85

15. Infrared Spectrograph of N- Benzyl- 2,5- Dime thoxyb enzamide ...... 87

16. Infrared Spectrograph of N - ^ - Phenylethyl- 2.5- Dimethoxybenzamide ...... 88

17. Infrared Spectrograph of N- X - (4- Morpho- lino) propyl- 2,5- Dimethoxybenzamide Hydrochloride...... 89

18. Infrared Spectrograph of N,N- Diisobutyl- 2.5- Dimethoxybenzamide • 92

19. Infrared Spectrograph of N,N- Diisoamyl- 2,5- Dimethozyb enzamide ...... 95

20. Infrared Spectrograph of N,N- Dibenzyl- 2,5- Dimethoxybenzamide ...... 94

21. Infrared Spectrograph of N- (2,5- Dimethoxy­ benzoyl) Piperidine ...... 95

22. Infrared Spectrograph of N- (2,5- Dimethoxy­ benzoyl) Pyrrolidine ...... 96

23. Infrared Spectrograph of 2,5- Dimethoxybenzoyl u r e a ...... 99

24. Plot of the Log of the Distribution Coeffi­ cients (log £) vs. p H ...... 108

vlli INTRODUCTION

Salicylic acid and sodium salicylate have been employed as analgetics for many years. In the body, salicylic acid is biologically oridized to gentisic aoid.

Gentisic acid (2,5- dihydroiybenzoio acid) has also been shown to possess analgetic and antipyretic properties

(1, 2, 3, 4).

Salicylamide is also used as an analgetic and anti­ pyretic, and in more recent years gentisic aoid amide has been shown to possess analgetic and antipyretic properties similar to salicylamide and salicylic acid (5, 6 , 7).

The relationship among these compounds can be seen from the following structural formulae:

HO-

7 \ \ c ' OH 7 \\/oH

salicylic acid gentisic aoid

NHi

salioylamlde gentisamide 2

Since both salicylic acid bûA gentisic acid as well as their simple amides possess analgetic properties, this investigation was directed toward the preparation and study of some derivatives of gentisic acid in the fozm of

N- substituted amides. By using different amines that have been shown to possess analgetic or depressant action either alone or in chemical combination with other compounds that do possess such activity, it is hoped that some new important compounds can be added to the presently available analgetics. DISCUSSION OP THE LITERATURE

General

Although salicylic acid has been used for the

treatment of rheumatic conditions since 1376 and sodium

salicylate since 1877, gentisic acid, the biological

oxidation product of salicylic acid (8 ), was not used medicinally until 1948, In that year Meyer and Ragen (1)

salicylic acid gentisic acid

introduced gentisic acid for the treatment of acute rheumatic conditions, after observing that salicylic acid

inhibited hyaluronidase in vitro only in very high concen­ trations whereas gentisic acid inhibited hyaluronidase in vitro in concentrations of a few micro grams per cubic centimeter, Gentisic acid was used in five patients with acute rheumatic fever and in seven patients with rheumatoid arthritis. Prompt and effective control of these disorders was noted, 3 4

Other workers have also shewn that gentisic aoid

possesses antiarthritic and analgetic properties similar to

salicylic aoid (£, 3). Schaefer et al. (4) and Clarke ^

al. (9) have shewn that 2,5- dihydroxyhenzoic acid produces

prompt and as effective control of the clinical manifesta­

tions of acute rheumatic fever as does salicylic aoid and

sodium salicylate. On the other hand, Rosenberg et al,

(10) and Batterman et al, (11) found only transient

improvement in acute rheumatic fever with this compound.

Sodium gentisate has been shown to be less toxic than sodium salicylate (12), having an LD50 equal to 3,1

grams per kilogram intraperitoneally in rats as compared with an ID^q of 0 , 8 8 grams per kilogram for sodium salicylate. Orally sodium gentisate was tolerated up to 5 grams per kilogram and sodium salicylate only 2,2 grams per kilogram in rats.

By means of labeled salicylic acid it has been found that gentisic acid is excreted in the urine to the extent of only 4 to 5 per cent of the original salicylic acid. In spite of the low percentage, the action of salicylic aoid is believed to be due to this biological oxidation product end not to the salicylic aoid as such (1 ,

13, 14), Christian and Crabtree (15), by using aspirin

(acetylsalicylic acid) with the free carboxyl group labeled with C^^, attempted to prove that the action of aspirin and 5 salioylio aoid is dependent upon the gentisic acid produced biologically. Aspirin with the labeled carbon was injected into rats in which fever had been produced by feeding with peptone solutions* After forty-five minutes the blood, lungs, and adrenal and pituitary glands were examined.

These organs are known to accumulate aspirin. The pituitary gland was found to contain labeled gentisic aoid, but none of the other organs shewed any of the labeled gentisic acid. Thus the site of the oxidation is believed to be the pituitary gland.

The knowledge of the effect of salicylic acid on hyaluronidase in rheumatic conditions dates back to 1946

(16, 17), Guerra believed that rheumatic fever is a disease of the mesenchymal tissue. During the evolution of the disease, which may be bacterial or endogenous in origin, there is a change in the permeability of this tissue pointing to the relationship of several character­ istics of rheumatism and the spreading factor in connective tissue.

Meyer and Palmer (18) established the importance of the spreading effect of hyaluronidase on connective tissue*

They pointed out that hyaluronic acid is the principal substrate of connective tissue and of mucoid structures, which are the main structures affected in rheumatism, exam­ ples being the articulations and the synovial membranes. 6

Guerra shewed that hyaluronidase of bacterial or

testicular origin increases the spreading of Ewans Blue in

humans and spreading India Ink in rabbits. This spreading was attributed to an enzymatic activity which hydrolyzed hyaluronic acid, the principal substrate of hyaluronidase.

Since viscosity of the extracellular fluids decreased, the passage of fluids, exudates, and pathogens was favored.

Sodium salicylate when given orally or intravenously caused a 57 per cent reduction of the diffusion of India Ink in the skin of rabbits. This diffusion was attributed to the effect of the hyaluronidase. When humans with rheumatic fever were given intradermal injections of a mixture of hyaluronidase and Evans Blue, enormous diffusion of the dye resulted. In addition pronounced local edema was noted.

Salicylates given in these cases inhibited the spreading of the dye in connective tissue and reduced localized edema,

Guerra, therefore, believed that the activity of the salicylates in rheumatic conditions was due to the inhibitory effect on hyaluronidase. However, it has been demonstrated that salicylates do not inhibit hyaluronidase in as low concentrations in vitro as they do in vivo (19,

20), Swyer (21), by using the visoosimetrie technique in vitro, recorded that sodium salicylate and acetylsalicylic aoid do not inhibit hyaluronidase except in relatively high concentrations; i.e., in excess of 3 per cent and 0,33 per 7 oent, respectively. At these concentrations, the inhibitory activity was attributed to a lowering of the pH, and in the case of the sodium salicylate, to an increase in the salt concentration. Thus Swyer believed that the results obtained by Guerra were due to the presence of histamine or some substance of similar action, and that sodium salicylate, by inhibiting the activity of this sub­ stance, reduced the apparent spreading effect caused by hyaluronidase.

Meyer and Regan (1, 13) believed that the inhibitory action of salicylates was due to the biological oxidation product, gentisic aoid, and not to the salicylates as such.

However, Roseman et al, (22) shewed that pure gentisic acid was devoid of antihyaluronidase activity, but that crude gentisic aoid or gentisic acid treated with alkali in the presence of air did shew such activity. Similar results were obtained with 2,5- dihydroxyphenylacetic aoid, com­ monly known as homogentisio acid. Therefore they concluded that the activity of gentisic aoid was probably due to impurities in the product which had been formed by oxida­ tion, Roseman (23) again in 1952 showed that analytically pure gentisic aoid did not inhibit hyaluronidase in vitro but that the active substance was a polymerization product of the oxidized quinone, identified as *^humic aoid," 8

Hcwever, the structure of this aoid is unknown. Other reports have also shown the lack of inhibitory power of gentisic acid for hyaluronidase (24, 25).

Guerra also showed that the amount of glucuronic aoid excreted in the urine increased following salicylates.

However, when gentisic aoid was used in place of sali­ cylates, the urinary concentration of glucuronic acid decreased. Since hyaluronic acid is composed of D- glu­ cosamine, glucuronic aoid and N- aoetygluoosamine, the antiarthritic action of gentisic acid appeared to be suit­ able in respect to excretion.

Another cause of rheumatic conditions is believed to be due to hormonal imbalance. The salicylates may stimu­

late the adrenal cortex causing a liberation of

17- ketosteroids. In rats and guinea pigs cortical stimu­ lation by the salicylates can be noted by an increase of

17- ketosteroids in the urine with an accompanying decrease in circulation eosinophils (26). However, administration of salicylates to four medical students failed to show any decrease in the circulating eosinophils.

Thiebst et al. (27), in an attempt to correlate rheumatoid arthritis and permeability of the synovial mem­ brane, showed that sodium salicylate was more effective in retarding this permeability than was sodium gentisate. The retardation was based on the intra-articular injection of 9 phenolphthalein and the recording of the time for the appearance of urinary coloration. This retardation has a favorable action in rheumatic conditions. The mechanism of action was believed to be associated with the function of the adrenal cortex.

The amide of salicylic aoid has been known for more than one hundred years, but only in recent years has it gained recognition as a potential analgetic agent. As early as 1891 Nesbitt (28) suggested that salicylamide might prove to be a worth-while substitute for the acid because of similarities in structure. However, early in the twentieth century, pharmacological investigations of this drug were only fragmentary. More complete investiga­ tions of this compound have been reported by others (29,

30, 31, 32). In 1950 k/eland (33) and Wegman (34), through their clinical reports, renewed the interest in salic yl­ amide as a potentially useful and safe analgetic agent.

Because of the gastric irritation attributed to the free carboxylic aoid function that is produced in many persons using salicylates, it was postulated that persons suscep­ tible to gastrointestinal irritation from salicylates might tolerate salicylamide, since this compound does not produce a free carboxylic acid in the digestive tract (35). When

Mandel et al. (36), in 1952, used salicylamide with labeled carbonyl, they presented convincing evidence to 10

support the view that salioylamlde was not hydrolyzed to

the free acid in the body, since almost all of the amide

was accounted for unchanged or as the ethereal glucuronide

in the urine.

The more recent phamacological studies of salioyl-

amide have shown that this amide, in analgetic and anti­

pyretic activity, is equivalent to or greater than the free

acid. Although the acute toxicities of the two drugs

appear to be about equal, the amide caused death by central

nervous system and respiratory depression rather than by

stimulation and convulsions characteristic of the acid.

Furthermore, the chronic and oral toxicity was far less than that of the free aoid, with gastrointestinal irrita­

tion less predominant.

It was observed by Bray et al. (37) and later con­ firmed by Becher et al. (38) that gentisamide is a metabolic product of salicylamide in the same way that

gentisic acid is a biological oxidation product of salicylic acid. The biological oxidation of salicylamide may be represented by the following reaction:

salicylic aoid amide gentisic acid amide 11 However, there has been no attempt to shew that salicylamide might be active through its biological oxida­ tion product. Nevertheless, such a postulation might be possible by correlating the similarities in pharmacological action and chemical structure between these two pairs of analgetic-antipyretic agents, i.e., salicylic acid and gentisic aoid; salicylamide and gentisic acid amide.

In view of the renewed interest in salicylamide as a potentially useful analgetic-antipyretic agent. Way et al.

(5) in 1953 studied the biochemorphology of eighty-one new derivatives of salicylamide in the hope of obtaining information that might lead to more potent and useful analgetics. The compounds investigated were based on the following structural formula:

(H,e^KjCO,

(H

The analgetic activity was measured according to the procedure set forth by Brodie, Way, and Smith (40), and an

ADgQ (the dose required to produce analgetic effects in 50 per cent of the animals) was determined for all eighty-one compounds. In addition to the ADgQ, the LD^q was also determined intraperitoneally in rats and thus a ratio of 12

IDgg/ADgQ, the therapeutic ratio, was obtained. All eighty-

one compounds had the ability to raise the pain threshold

of the animals, and most of the compounds appeared to be

central nervous system depressants, whereas some possessed

pronounced hypnotic properties. However, the hypnotic

dose was greater than the dose required to elevate the pain

threshold. No notation was made concerning which compounds

possessed this hypnotic activity.

Among the compounds tested by Vfay et al. were the

amide of gentisic acid and N- methyl gentisic aoid amide.

The LD50 gentisamide was found to be 900 mg./Kg., and

the AD5Q was 350 mg./Kg., giving a therapeutic ratio of

approximately three. In comparison, the LD50 o f salicyl­

amide was found to be 350 mg./Kg., and the ADgQ was 175

mg./Kg., giving a therapeutic ratio of two. The LDgQ of

N- methyl gentisic acid amide was found to be 600 mg./Kg.,

and the AD5 Q was found to be 200 mg./Kg. The therapeutic

ratio thus remained the same as tlie simple gentisamide.

The toxicity of salicylamide is decreased by

methylating the hydroxyl group to produce the methyl ether.

The LDgQ of the methyl ether was 450 mg./Kg., whereas the

AD50 decreased to 150 mg./Kg. to make the therapeutic ratio

increase to three. By the introduction of a methoxyl group

in the 5 position of salicylamide, the LDgQ of the result­

ing compound increased, accompanied by a decrease in the 13

ADgQ (LDgQ of 750 mg./Kg. and an ADgQ of 125 mg./Kg. ).

Thus the therapeutic ratio increased to six. The latter compound, 5- methoxysalicylamide, was less toxic and more active than salicylamide.

The most promising compound of the series tested by

Way et al. was N,N- dimethyl- 3- phenyl salicylamide, which had a toxicity of low magnitude and was the most potent analgetic (LDgQ of 700 mg./Kg. and an ADgQ of 60 mg./ Kg.). Two of the compounds with low orders of toxicity, N- ( p - hydroxyethyl)- 3- phenylsalicylamide and

3- phenylsalicylamide, were reported as being investigated clinically; however, no results of this investigation have been published.

In 1954 Earth on and Sigroth (6 ) reviewed the thera­ peutic value of gentisic acid amide and presented some of the chemical properties of this amide. The amide was prepared by the action of aqueous ammonia on an ester of gentisic acid. The urinary excretion was measured by ultraviolet spectrophotometric methods and shewed that 44 per cent of the amide was excreted in eight hours, with 61 per cent excreted in twenty-four hours. By paper electro­ phoresis, Barth on and Sigroth were able to demonstrate that the amide partially combines with the albumins of the plasma. 14

In olinioal trials, involving twenty persons with rheumatic fever, Bar thon and Sigroth found it difficult to obtain effective blood concentrations of gentisic acid amide, owing to the rapid excretion of the drug. However, they did point out that ordinary analgetic effects might not require as high blood concentrations. Thus it was reported that gentisic acid amide has a slight but unreli­ able antipyretic effect in rheumatic fever and that it is only mildly analgetic, definitely inferior to salicylamide or sodium salicylate. Nevertheless, no toxic or side effects were noted.

Preziosi in 1953, in a study of some new salicylic acid and gentisic acid derivatives in the hope of finding more potent antipyretics, found that the antipyretic action decreased among the compounds tested in the following order: salicylic hydrazide, aspirin hydrazide, N,N — diethyl salicylamide, gentisic acid amide, o- ethozy- benzamide, salicylanilide, diacetylgentisic acid, and

0 - diethylaminoethoxybenzamide. However,all of these com­ pounds had the ability to reduce body temperature in tnree to four hours in rabbits injected with pyrogens.

Although sodium salicylate and sodium gentisate have been reported to possess no antihistamlnic activity (1 2 ),

Isliker and Schonholzer (43) have demonstrated that gentisic aoid and gentisic acid amide have the ability to 15

give prolonged end effective protection to experimental

animals subjected to aerosol inhalation of histamine,

Hcwever, salicylic acid and salicylamide did not possess

this activity.

Some new gentisic aoid derivatives

Nash et aJL, (41, 42, 43) in 1953 prepared and

determined the LDgQ»s of some new gentisic aoid derivatives

in the form of esters. These eaters involved (1) the

carboxylic acid function; (2) the 5- phenolic hydroxyl

group; and (3) esters on both the 5- phenolic hydroxyl

group as well as the carboxylic aoid function. The esters

of the carboxylic acid function were of the dialkylamino­

alkyl variety (2- diethylaminoethyl and 3- diethylamino­

propyl) with or without substitution on the 5- phenolic hydroxyl. In general, the 2- diethylaminoethyl esters were less toxic than the 3- diethylaminopropyl esters. The com­

pounds in which the 5- phenolichydroxyl had been methylated proved to be the most toxic.

Estérification of the 5- phenolic hydroxyl with phenylacetic aoid, p- nit ob en zoic acid, anisic aoid, acetylsalicylic aoid, and succinic acid gave a series of

compounds, Nash tested these compounds for toxicity,

Moore et al, (44, 45) continued the work on esters

of gentisic aoid. Three of the compounds prepared by Moore 16 were 5,5- digentisyl phthalate, 5- diphenylaoetyl gentisic aoid, and 5- diethylaoetyl gentisic acid. These compounds are esters of the 5- phenylic hydroxyl of gentisic aoid with other carboxylic acids, Moore also prepared the fol­ lowing amino alcohol esters of the carboxylic aoid func­ tion: 2- diethylamino- ethyl- 5- ethoxy salicylate hydrochloride; 2 - n- butylaminoethyl gentisate hydro- chrloride; 2- dimethylaminoethyl gentisate hydrochloride; and 3- (1- methylpiperidyl) methyl gentisate hydrochloride.

The LDgQ»s of all the above compounds were deter­ mined, Some of the esters were screened for antispa amodie, local anesthetic, and for anticonvulsant activity.

Of the esters tested, 2- diethylaminoethyl- 5- ethoxy salicylate hydrochloride was shewn to be somewhat less active than papaverine hydrochloride as an anti- spasmodic, and produced motor anesthesia only slightly slower than procaine hydrochloride. None of the compounds screened for anticonvulsant action were sufficiently active to protect mice from a lethal dose of Metrazol,

Preparation of amides

In general there are seven methods by which amides may be prepared (46, 47).

1, From acyl halides and ammonia or amines

ROOX ♦ 2 NHR»2 — » R00NR*2 ♦ R ’gNH • HX. 17

This réaction goes at room temperature* Two moles

of the amine are necessary because of the inappreciable

dissociation of the amine halide at the temperature of the

reaction. It also is the most convenient method for the

preparation of amides when the solvent is ether or benzene.

In this method, it is usually not necessary to isolate the

acyl halide in pure form. However, when the acyl halide is

prepared by using PCI5 or thionyl chloride (8001g), the resulting POOlg or the excess SOClg should be removed before the final reaction with the amine, since one mole of

PCI5 would react with five moles of the amine and one mole of SOClg would react with two moles of the amine (47).

2. From acid anhydrides and ammonia or amines

(RCO)gO ♦ 2 HER*g — V RCONE»g ♦ NHR»g • RCOOH.

Although this reaction goes as indicated in the presence of an excess of ammonia or amine, it is possible to convert all of the base into an amide in the presence of an excess of the anhydride because of the ease of the dis­ sociation of the acid salt of the amine.

3. From esters and ammonia or amines

RGOOR» ♦NHR»*g — » RCONR»»g * R*OH.

This reaction is usually conducted at room tempera­ ture in an aqueous or alcoholic solution of the mine.

This reaction is analogous to the reaction of an ester with water (hydrolysis) or with an alcohol (alcoholysis) and is 18

tenaed ammonolysis* Hydrolysis or alooholysis requires

either basio or acidic conditions, the amine, in this case,

provides basic conditions favorable for the transformation*

4. From ammonium salts of oarboxylic acids by

thermal decomposition

NHR»2 • RCOOH — » RCONR’g 4- HgO.

Water is removed at the temperature of decomposition

of the ammonium salt, forcing the reaction to completion.

An excess of the carboxylic acid is used to minimize dis­

sociation of the ammonium salt.

5. From nitriles by the action of alkaline solutions

of hydrogen peroxide

RON 4. HgOg OH^ ROONHg f l/2 Og.

This reaction is suitable for the preparation of

simple amides. Because of the nature of the reactants, this method is not applicable to the preparation of

N- substituted amides.

6 . From other amides by acid exchange

ROONHg 4* R'COOH RCOOH 4. R ’OONHg.

When an amide is heated with another carboxylic

acid, an exchange reaction takes place which leads to an

equilibrium mixture of the two amides and the two acids.

However when urea (carbamide) is used as the starting

amide, the unstable carbamic acid results and the reaction

goes to completion. 19

RCOOH * COfHNglg — HgNCOOH 4. RGONHg.

HgNCOOïï — NH3 4- 00g.

7. Another method for the preparation of N- substituted

amides involves the reaction of an alkyl halide

with the sodium salt of the amide, usually obtained

by the action of sodamide (NaNHg) on an amide.

RCONHg f NaNHg — ► RCONHNa t- NH3 .

RCONHNa R»Z — # ROONHR^ 4- NaZ.

The reaction of the amide with sodamide is conducted in benzene or anhydrous liquid ammonia.

After alkylation, the monosubstituted amide can be converted into its sodium derivative and this, in turn, may be alkylated to give a disubstituted- amide.

In addition to the alkyl amide derivatives obtained by this reaction, imino ethers (RC(;NE)OR) also are formed.

Thus a mixture of final products is obtained.

Of the seven general methods described for the preparation of amides, the reaction which involves acid halides and amines was selected for the synthesis of some

N-substituted amides of gentisio acid. The dimethyl ether of gentisio acid (2,5- dimethoxybenzolc acid) was used as the starting acid instead of gentisio acid in this study for the following reasons:

1. The resulting N- substituted amides would be more stable molecules. They would be less susceptible to 20

oxidation than N- substituted amides of gentisio aoid* If

one examines the gentisio aoid molecule, it is noted that

the two phenolic hydroxyls are para to each other. Thus they are capable of being oxidized to a paraquinoid struc­ ture. Methoxyl groups para to each other are not suscepti­ ble to such an oxidation.

2. There is evidence to show that méthylation of a phenolic hydroxyl group or éthérification of a phenolic hydroxyl group tends to decrease the toxicity of the parent phenolic compound. Way et al. (5) showed that the LDgQ of salicylamide increased or the toxicity decreased by the formation of the methyl ether of the phenolic hydroxyl.

3. Way et al. (5) have shown that N- substituted amides of salicylic acid methyl ether, as well as

5- metfaoxysalicylamide, possess analgetic activity. GENERAL PROCEDURES

1. All melting points were determined on a Pisher-

Johns Melting Point Apparatus, 115 volts, 50 - 60 oyole

A. 0., and are reported unoorreoted.

B, Nitrogen determinations were made by the

Galbraith Micro analytical Laboratories, 103 W. Fifth Ave.,

Khoiville 17, Tennessee.

3. Infrared spectrographs were prepared in The Ohio

State University Department of Chemistry on a Baird

Associates, Inc., Recording Infrared Spectrophotometer,

Model B. The infrared spectrographs of the liquid products were prepared by using the pure liquid compounds, and the infrared spectrographs of the solids were prepared from samples made by triturating the compound (approzimately 1 mg.) with potassium bromide (approzimately 400 mg.) and compressing the triturates into pellets by using a pressure of 2 0 , 0 0 0 pounds per square inch.

4. Several of the N- substituted amides prepared were isolated as hydrochloride salts of an amino function.

The molecular weights of these compounds were determined by the Mohr titration of the chloride ion in aqueous solution

21 22 with standard silver nitrate and potassium ohromate as the indicator (48). An average of two determinations are reported.

5. The optical properties of the crystalline products were obtained with a Spencer Pétrographie Micro­ scope. EXPERIMENTAL

Preparation of 2.5- dimetboiy- benzoioaoid

W.co

A modification of the method used by Mauthner (49) was used for the preparation of 2,5- dimethoxybenzoic acid.

Gentisio acid (50 gm. ) was dissolved in a solution of 60 Gm, of sodium hydroxide in 300 ml. of water. The resulting dark red-brown solution was cooled in an ice bath to about 10°G. To this mixture, 50 ml. of dimethylsulfate

(practical grade) was added and the mixture was stirred vigorously for 30 minutes while being cooled in an ice bath. An additional 50 ml. of dimethylsulfate was added and the stirring was continued for fifteen minutes. The reaction mixture was then heated under reflux for two hours. After refluxing, 10 Gm. of sodium hydroxide was added and the mixture was again refluxed for two hours.

After cooling the reaction mixture, now a lighter red color, to room temperature, 30 Gm. of sodium hydroxide

23 24

was added, and the reaction was further cooled in an ice

bath to 10°C. Again 50 ml, of dimethylsulfate was added,

and the mixture was stirred for 30 minutes. A second 50

ml, of dimethylsulfate was added and the mixture was

stirred an additional 15 minutes. The resulting mixture

was then refluxed for two hours, following which 20 Gm, of

sodium hydroxide was added and the refluxing was continued

for an additional two hours. The reaction mixture was then

placed in the refrigerator at 0°C.

The cold solution was poured slowly, with constant

stirring, into 200 ml, of 10 per cent hydrochloric aoid,

producing a heavy precipitate. After stirring at room temperature for 30 minutes, the brownish-white precipitate was filtered by suction and dried in a vacuum desiccator

over P2 O5 .

The resulting brownish-white product was dissolved

in 2 0 0 ml, of benzene, giving a deep yellow-brown solution.

This solution was decolorized with charcoal and filtered while hot through a fluted filter. Petroleum ether (30-60) was added to this hot almost colorless solution until a

slight turbidity appeared. White needle-like crystals

separated on cooling, which were removed by suction filtra­ tion and dried in a vacuum desiccator over PgOg, The dried crystals weighed 53 Gm, (90 per cent based on the weight of

gentisio acid used). This compound made a melting point of 25

77° to 78®C. wbioh is in agreement with the melting point reported by Mauthner (49), In addition an alcoholic solu­ tion of the compound gave no color with ferric chloride test solution, thus indicating the absence of phenolic hydroxyls.

Reactions:

HfO

COH V \Va-c OCH,

ON OCH,

gentisic acid methyl 2,5- dimethozybenzoate

H,Co NaOW COCH ON

methyl 2,5- sodium 2,5- dime th Qzyb en zoat e dime th ozyben zoat e

H / 0 H3C 0

/ W c OH

OCH, OCH,

sodium 2 ,6 - 2,5- dimethoxy- dimethoxybenzoate benzoic aoid 26

Preparation of 2,5- aimetbozy- benzoyl chloride

HjCO; '/ \\/c, ocHj

The acid chloride of 2,5- dimethoxybenzoio aoid was

originally made by lîauthner (49) by using equal molar quan­ tities of 2,5- dimethoxybenzoio aoid and POI5 . However, this reaction took place violently and was difficult to control. In addition it was difficult to remove any excess

PCI5 , Any excess of POI5 remaining after the reaction would interfere with the subsequent reaction with the amine. A more suitable method for the preparation of the acid chloride involves the use of thionyl chloride (SOOlg),

The by-products of the reaction are gaseous (SOg and HOI), and any excess SOClg could be removed readily by distilla­ tion.

To prepare the aoid chloride of 2,5- dimethoxy­ benzoio aoid, 9.11 Gm. (0.05 mole) of the aoid was dis­ solved in 25 ml. of dry benzene. To this solution, 0.1 mole (11.9 Gm.) of thionyl chloride was added, producing a deep yellow solution. The mixture was heated under a reflux condenser, fitted with a drying tube, until it was s? no longer possible to detect the evolution of HCl with ammonia test solution. The time required for this reaction was about four hours. Subsequent reactions for the syn­ thesis of additional amounts of the aoid chloride were run for four and one-half hours.

After the required time for heating, the excess

SOClg and the solvent were removed by vacuum distillation with a water pump and a minimum of heat. In order to assure complete removal of the excess SOClg, an additional

25 ml, of dry benzene was added and the distillation was continued until a yellowish oily residue remained in the reaction flask.

Since it is not necessary to isolate the pure acid chloride for the subsequent reaction with the amines (47), this yellowish oily product, 2,5- dimethoxybenzoylchloride, was not further purified.

Classification of N- substituted amides prepared

As mentioned earlier, 2,5- dimethoxybenzoio acid was selected as the fundamental aoid for the preparation of a series of N- substituted amides corresponding to the fol­ lowing general structural formula. 28

The amides to be prepared may be classified as follows :

I. Monosubstituted amides (R* - H)

A. R = Alkyl

B. R r Dialkylaminoalkyl

C. R « Alioyolio

D. R = Aromatic

S. R - Aralkyl

F. R s HeteroQTOlic alkyl

II. Disubstituted amides (R s R*)

A. R and R» = alkyl

B, R and R» r aralkyl

III. Miscellaneous

The general reactions to be used for the preparation of these compounds are as follows:

W/Oy HjCo- y W/,

2,5- dime thoiyb an zoic aoid 2,5- dimethozybenzoyl chloride 89

C C I - h M L

OCH,

2,5- dimethoxybenzojl N- substituted amide chloride of 2,5- dimethoxy­ benzoio acid

Monosubstituted amides of 2.5- dimethoxybenzoio acid

Alkyl

1. N- n- propyl- 2,5- dimethoxybenzamide

/? H C-N-

To prepare N- n- propyl- 2,5- dimethoxybenzamide, the acid chloride of 2,5- dimethoxybenzoio aoid (0.05 mole or 10.03 Gm.) was prepared with thionyl chloride as pre­ viously described and was dissolved in 25 ml. of anhydrous ether. The ether solution of the aoid chloride was added dropwise to a cold ether solution of n- propyl amine, (0 . 1 mole or 5.91 Gm.) in 25 ml. of anhydrous ether. During the addition of the aoid ahloride, the amine solution was stirred oontinuously in an ioe bath. After the addition of 30

the aoid chloride, the mizture was stirred at room temper­

ature for 30 minutes to assure complete reaction.

The reaction mizture was transferred to a separatory

funnel and eztracted with 25 ml, of 10 per cent hydro­

chloride to remove any unreacted n- propylamine. Twenty-

five ml. of 5 per cent aqueous sodium bicarbonate were

added to remove any unreaoted 2,5- dimethozybenzoic acid.

The resulting yellcw ether solution was removed and dried

over anhydrous sodium sulfate.

The dry ether solution was filtered and the ether was removed in a current of dry air passing over the solu­ tion. A yellow oily product resulted, which weighed 10.5

Gm, Attempts to crystallize the product by low temperature crystallization or by trituration with petroleum ether, in which the substance was insoluble, were unsuccessful. The product was distilled under reduced pressure. A colorless

5 Gm, fraction which boiled at 163? to 163°C. at 2 mm, of

Hg was collected. The yield was 45,4 per cent, based on the weight of the 2,5- dimethozybenzoic aoid used.

Percentage nitrogen for GigH^yNOg:

calculated 6.27

found 6.31

Refractice indez

1.5420 31 2, N- n- butyl- 2,5- dimethozybenzamide

H c 0

OCH.

N- n- butyl- 2,5- dimethozybenzoio aoid was prepared according to the procedure used for the preparation of the

N- n- propyl derivative by using 0.1 mole (7.32 Gm.) of n- butylamine and 2,5- dimethozybenzoyl chloride obtained from 9.11 Gm, (0.05 mole) of 2,5- dimethozybenzoic acid.

An orange-brown oily product weighing 9,8 Gm, was obtained. This produce also failed to crystallize or solidify. A 4.75 Gm, oily fraction, which boiled from 168° to 169°C. at 2 mm, of Hg, was collected. The yield was 40 per cent, based on the weight of the 2,5- dimethozybenzoio acid used.

Percentage nitrogen for

calculated 5.90

found 5.87

Refractice indez

1,5385 52 5. N- isoamyl- 2,5- dimethoxybenzamide

H ro

CH

The starting materials for the preparation of

N- isoamyl- 2,5- dimethoxybenzamide were 2,5- dimethoxy- benzoyl chloride (0.05 mole), obtained from 9.11 Gm. (0.05 mole) of 2,5- dimethoxybenzoio aoid, and 0.1 mole (8.72

Gm, ) of isoamylamine. The procedure used for the prepara­ tion of this derivative was the same procedure employed for the preparation of the N- n- propyl and the N- n- butyl derivatives.

An oily orange colored product was obtained. This product weighed 11.2 Gm. Since this product would not crystallize or solidify, it was distilled and the fraction which boiled at 186° to 188°C. at 2 mm. of Hg was collected.

The weight of this oily product was 9.3 Gm., or 75.9 per cent of the theoretical. It was slightly yellow in color,

percentage nitrogen for C1 4 H21NO3 : calculated 5,57 found 5.47

Refractive index

ngO 1,5330 33

Dialkylaminoalkyl

1. N - y - dimethylaminopropyl- 2,5- dime thoiyb enza-

mide hydrochloride

H X o

/ CH,CH, CH, N Hci

N - Y - dimethylaminopropyl- 2,5- dimethoxybenzamide hydrochloride was prepared by reacting dimethylaminopropyl- amine with 2,5- dimethoxybenzoyl chloride. The acid chloride of 2,5- dimethoxybenzoio acid was prepared from

9,11 Gm, (0,05 mole) of 2,5- dimethoxybenzoio aoid, as pre­ viously described, Dimethylaminopropylamine (0,05 mole or

5.06 Gm,) in 100 ml, of dry ether was added dropwise to the acid chloride in 50 ml, of dry ether with constant stirring,

A white precipitate resulted, which was filtered at the completion of the reaction, washed with ether, and dried in a vacuum desiccator over PgOg,

The dry product was dissolved in a minimum amount of hot absolute ethanol. This solution was then boiled with charcoal and filtered while hot. Anhydrous ether was added to the hot filtrate until a slight turbidity resulted.

Upon cooling, white needle-like crystals were obtained which, when filtered and dried in a vacuum desiccator over 34

PgOS; weighed 13,4 Gm. (89,5 per cent yield based on the weight of 2,5- dimethoxybenzoio aoid used). The crystals melted at 163° to 164°C,

The product was water soluble; an aqueous solution gave a positive test with AgNOg test solution, indicating the presence of chloride ion. It also gave an oily pre­ cipitate when made alkaline with 5 per cent NaOH, indicating that the product was an amide, since the amine itself is water soluble. The solid gave a positive Beilsteintest for chlorine.

Percentage nitrogen for C^^EggNgOgCl:

calculated 9,25

found 9,15

Molecular weight determination by the Mohr

titration:

calculated 302,8

found 301,2

2, N-y - diethylaminopropyl- 2,5- dimethoxyben­

zamide hydrochloride

HCl 35

N-y - diethylaminopropyl- 2,5- dimethoxybenzamide

hydrochloride was prepared according to the method described

for the preparation of the N - / - dimethylaminopropyl

derivative, by the reaction of 7,45 G-m. (0.05 mole) of diethylaminopropylamine with 2,5- dimethoxybenzoyl chloride, obtained from 9.11 Gm. (0.05 mole) of 2,5- dimethoxybenzoic aoid. The white product obtained follow­ ing the reaction was dried in a vacuum desiccator over

^2*^5* The dry product was then dissolved in a minimum amount of hot absolute ethanol. This solution was boiled with charcoal end filtered while hot. Anhydrous ether was added to the hot filtrate until a slight turbidity appeared, White needle-like crystals which separated on cooling were filtered and dried in a vacuum desiccator over

PgOg. The dried crystals weighed 14.8 Gm. and had a melt­ ing point of 169° to 170°C. The yield was 90 per cent of the theoretical amount based on the weight of 2,5- dimethoxybenzoio aoid used. This product gave all the same tests described for the dimethylaminopropyl derivative.

Percentage nitrogen for C^gHg^NgOgCl:

calculated 8,47

found 8,45 36

Molecular weight determination hy the Mohr

titration;

calculated 330.8

found 329.9

Alieyolio

1. N- oyolohezyl- 2,5- dimethoxybenzamide

For the preparation of N- oyclohexyl- 2,5- dimethoxy­ benzamide, the acid chloride of 2,5- dimethoxybenzoio aoid was prepared by the action of thionyl chloride on 0,05 mole

(9.11 Gm. ) of 2,5- dimethoxybenzoio acid, as previously described. The aoid chloride was dissolved in 25 ml, of anhydrous ether. The ether solution of the aoid chloride was added dropwise, with constant stirring, to 0 . 1 mole

(9,92 Gm,) of oyolohexylamine in 25 ml, of anhydrous ether.

The reaction was kept in an ioe bath during the addition of the acid chloride. A white preoipate resulted. After the addition was completed, the reaction mixture was stirred at room temperature for 30 minutes, transferred to a separatory funnel, shaken with 50 ml, of 10 per cent hydrochloric aoid to remove any unreaoted amine and then 37 with 50 ml. of 4 per cent sodium bicarbonate to remove any

unreaoted aoid chloride. The final ether solution was washed with water and then dried over sodium sulfate.

The ether was evaporated* leaving a yellowish

colored oily product which solidified upon standing at room

temperature overnight. This solid product was recrystal­

lized several times from petroleum ether (30-60), using

charcoal as a decolorizing agent. The final recrystal- lieation yielded needle-like crystals which were still slightly yellcw in color. After drying in a vacuum over

PgOg, the crystals weighed 10.6 Gm. (81 per cent of the theoretical) and had a melting point of 85° to 8 6 °C.

Percentage nitrogen for C-j^^Ng^NO^:

calculated 5.32

found 5.18

Aromati o

1. N- 2’- methylphenyl- 2,5- dimethoxybenzamide

H 3C 0

/ \ \ l u / V

The starting reagents for the preparation of N- 2’- dimethylphenyl- 2,5- dimethoxybenzamide were 2,5- dimethoxy­ benzoyl chloride, obtained from 9.11 Gm. (0.05 mole) of 38

2,5- dimethozytienzoio aoid, and 10.72 G-m, (0.1 mole) of

0 - tduidine.

The acid chloride was dissolved in 25 ml, of anhydrous ether. This ether solution of the aoid chloride was added dropwise with constant stirring to 25 ml, of anhydrous ether containing the o-toluidine, A white pre­ cipitate resulted. After the addition of the acid chloride was completed, the reaction mixture was stirred at room temperature for 30 minutes to assure complete reaction.

The ether was then evaporated and the residue was washed with 50 ml, of 10 per cent hydrochloric aoid to remove any unreaoted amine and was then filtered. The resulting yellow-white product was washed with 5 per cent aqueous sodium bicarbonate to remove any unreacted acid chloride and then was washed with water, ^he washed product was dissolved in ethanol. The resulting yellow solution was boiled with charcoal and filtered while hot. Water was added to the colorless filtrate until a slight clouding resulted. On cooling, white needle-like crystals were obtained which, after drying in a vacuum desiccator over

PgOg, weighed 11,5 Gm, (84,5 per cent of the theoretical) and had a melting point of 107° to 108°0,

Percentage nitrogen for Cj^gHiyNOg:

calculated 5,16

found 5,19 39 2. N- 3’- methylphenyl- 2,5- dimethosyhenzamide

C-N

OCH,

N- 3 ’- methylphenyl- 2,5- dimethoxybenzamide was prepared aocording to the procedure employed for the preparation of the N- 2’- methylphenyl derivative by using

2,5- dimethoxybenzoyl chloride, obtained from 0.05 moles

(9,11 Gm. ) of 2,5- dimethoxybenzoio aoid, and 0.1 mole

(10.72 Gm.) of m-toluidine.

The crude product was dissolved in ethanol and the resulting solution was boiled with charcoal and filtered.

Water was added to the hot filtrate until a slight clouding resulted. On cooling an oily product separated out, which solidified on further cooling. This solid mass was redis­ solved in ethanol and filtered. The alcoholic solution was heated to about 50°C. before the water was added to obtain turbidity. This time, white needle-like crystals separated on cooling to room temperature. These were filtered out by suction and dried in a vacuum desiccator over PgOg.

The dried crystalline product weighed 10.9 Gm, (80 per cent of the theoretical) and had a melting point of

63.5° to 64,5°0, 40

Peroentage nitrogen for C16H17NO5 :

calculated 5,16

found 5,14

3, N- 4»-methylphenyl- 2,5- dimethoxybenzamide

H.CO

N- 4»- methylphenyl- 2,5- dimethoxybenzamide was prepared by the previous method, employing the acid chloride obtained from 9,11 Gm, (0,05 mole) of 2,5- dimethoxybenzoyl chloride and 10,72 Gm, (0,1 mole) p-toluidine.

The white crystalline product obtained after recrystallization from an ethanol-water mixture was dried in a vacuum desiccator over PgOg, and weighed 11 Gm, (84,5 per cent of the theoretical). It had a melting point of

100° to 101°C,

Percentage nitrogen for C1 6 H1 7 NO3 :

calculated 5,16

found 5,22 41

4, N- 2',6 '- dimethylphenyl- 2,5- dimethoxybenza-

mide

W3CO7 \ H,C

OCH) HjC.

N- 2',6 *- dimethylphenyl- 2,5- dimethoxybenzamide

was made by the method described above by using 0 . 1 mole

(12,12 Gm.) of 2,6- xylidine and 2,5- dimethoxybenzoyl

chloride obtained by the reaction of SOClg with 9,11 Gm,

(0,05 mole) of 2,5- dimethoxybenzoio acid.

The white needle-like crystalline product obtained,

following recrystallization from a mixture of ethanol and water and drying over P2O5 as before, weighed 12 Gm. (84,5

per cent of the theoretical) and had a melting point of

124® to 125®C.

Peroentage nitrogen for 0 i7% g N 0 g:

calculated 4.91

found 5,02

5, N- 2 *,5*- dimethylphenyl- 2,5- dimethoxybenza- mide H3CO y \)-f.üY/ V

ÔCH,3 42

N- 2 ’,5»- dimethylphenyl- 2,5- dimethoxyhenzamide was also prepared according to the procedure already described by using 2,5- dimethoxybenzoyl chloride, obtained from 0.05 mole (9.11 Gm.) of 2,5- dimethoxybenzoio aoid, and 12.12 Gm. (0.1 mole) of 2,5- xylidine.

The crystalline compound was dried over PgOg in a vacuum, following recrystallization from a mixture of ethanol and water. The yield was 1 1 . 6 Gm,, or 81.5 per cent of the theoretical yield, and had a melting point of

117° to 1180c.

Percentage nitrogen for C1 7 H1 9 NO3 ;

calculated 4.91

found 5.00

6 . N- 2',4*- dimethylphenyl- 2,5- dimethoxy­

benzamide

H3C 0

CH,

0 CH3

K- 2%4*- dimethylphenyl- 2,5- dimethoxybenzamide was also prepared by the method described by using the acid chloride, obtained from 9,11 (0,05 mole) of 2,5- dimethoxy­ benzoio aoid, and 12,12 Gm. (0.1 mole) of 2,4- xylidine. 43

Following reorystallization from a mixture of ethanol and water, a white crystalline product was obtained which, when dried in a vacuum desiccator over PgOg, weighed 12 Gm.

(84,5 per cent of the theoretical) and had a melting point of 124.50 to 1260c.

Percentage nitrogen for CiyH^gNOg:

calculated 4,91

found 4,85

7, N- p- diethylaminophenyl- 2,5- dimethoxy-

benzamide hydrochloride

m

To prepare N- p- diethylaminophenyl- 2,5- dimethoxy- benzamide hydrochloride, 2,5- dimethoxybenzoyl chloride was prepared from 9.11 Gm. (0.05 mole) of 2,5- dimethoxybenzoio acid and SOClg, The acid chloride was dissolved in 50 ml. of anhydrous ether. The ether solution of the acid chloride was cooled in an ice bath, and 1 0 0 ml. of anhydrous ether containing 0.05 mole (8,21 Gm.) of diethyl- p- phenylene diamine was added dropwise with constant stirring.

After the addition of the diamine had been completed, a 44 yellow-white precipitate was present. To assure complete reaction, the reaction mixture was stirred at room tempera­ ture for 30 minutes. The precipitate was filtered by suction and washed with ether. The resulting product was recrystallized twice from a mixture of absolute ethanol and anhydrous ether, yielding 13,7 Gm. (75 per cent) of a white crystalline product which had a melting point of 163° to

164.5°C.

The product was water soluble, and an aqueous solu­ tion gave a yellow-white precipitate when made alkaline with 5 per cent NaOH. In addition, an aqueous solution gave a positive test with AgNOg test solution, indicating the presence of chloride ion. The solid gave a positive

Beilstein test for chlorine.

Percentage nitrogen for C1 9 H2 5 N 2 O3 CI;

calculated 7.68

found 7.54

Molecular weight determination by the Mohr

titration:

calculated 364.9

found 561,4

Aralkyl

1. N- benzyl- 2,5- dimethoxybenzamide

The starting intermediates for the preparation of

N- benzyl- 2,5- dimethoxybenzamide were 0.1 mole (10.72 On.) 45 H.CO / \vi5.c..y/ V

of benzylamine and 0.05 mole (10.03 Gm.) of 2,5- dimethoxy- benzoyl chloride. This acid chloride was obtained from

9.11 Gm. (0.05 mole) of 2,5- dimethoxybenzoio acid and was

dissolved in 25 ml. of anhydrous ether. This ether solu­ tion was added dropwise to a cold solution of 0 . 1 mole

(10.72) of benzylamine in 25 ml. of anhydrous ether, with

constant stirring, A white precipitate resulted. After stirring at room temperature for 30 minutes to assure com­ plete reaction, the ether was evaporated and the mixture was washed with 25 ml. of 10 per cent HCl to remove any unreacted amine. This resulted in a yellowish solid pro­ duct, which was filtered by suction, washed with 5 per cent

NaHCOg to remove any unreacted acid, and dried in a vacuum desiccator over PgOg. An attempt to recrystallize the product from alcohol and water yielded an oily product.

The product was recrystallized from a mixture of ether and petroleum ether (30-60), and charcoal was used as a decolorizing agent. A whits crystalline product was obtained, which was filtered by suction and dried over PgOg in a vacuum desiccator, The dried product weighed 10 Gm. 46

(73.5 per cent of the theoretical) and had a melting point of 90.5° to 92°C.

Percentage nitrogen for O1 6 H17NO3 :

calculated 5.16

found 5,25

2, N-^ - phenylethyl- 2,5- dimethozybenzamide

H/ro

N-^ - phenylethyl- 2,5- dimethoiybenzamide was pre­ pared by the reaction of 2,5- dimethoxybenzoyl chloride, obtained from 0.05 mole (9,11 Gm.) of 2,5- dimethoxybenzoio acid, with 0.1 mole (12.12 Gto.) of p- phenylethylamine.

The procedure employed for the preparation of the N- benzyl was used here. However, when the ether was evaporated, following the reaction of the acid chloride with the amine, a yellow oily product was present in addition to a white precipitate. In order to remove any unreacted p- phenyl­ ethylamine, 25 ml. of 10 per cent hydrochloric acid was added, and this product was extracted with 25 ml. of ether.

The ether extract was shaken with 25 ml. of 5 per cent aqueous sodium bicarbonate in a separatory funnel to remove 47 any unreaoted acid chloride. The ether layer was dried over anhydrous sodium sulfate.

Following filtration, the ether was evaporated to yield 13 Gm, of an orange oil, which solidified on standing at room temperature for about one week.

Attempts to recrystallize this orange solid from several solvent pairs failed to give a crystalline product.

Thus, distillation was employed as a means of purification.

The fraction which distilled at 238° to 239°C. at 2 mm, of

Hg was collected. The weight of this yellcw oily fraction was 8,2 Gm, or 57,6 per cent of the theoretical yield, based on the weight of 2,5- dimethoxybenzoio acid used.

Percentage nitrogen for OiyHigNOg:

calculated 4,91

found 4.93

Refractive index:

ngO 1,5825

Heterocyolic alkyl

1, N - Y - (4- morpholino) propyl- 2,5- dimethoxy­

benzamide hydrochloride

ÛCH3 48

The starting materials for the synthesis of N-Y -

(4- morpholino) propyl- 2,5- dimethoxybenzamide hydro­ chloride were 0.05 mole of 2,5- dimethoxybenzoyl chloride, obtained from 0.05 mole (9.11 Gm. ) of 2,5- dimethoxybenzoio acid, and 0.05 mole (7.2 Gm,) of N- (3- aminopropyl) morpholine. The acid chloride was dissolved in 50 ml. of anhydrous ether and cooled in an ice bath. The amine was dissolved in 1 0 0 ml. of anhydrous ether and added dropwise to the solution of the acid chloride, with constant stir­ ring. A white precipitate resulted. After the addition of the amine had bean completed, the reaction mixture was stirred at room temperature for 30 minutes to assure com­ plete reaction. The precipitate was filtered, washed with ether, and dissolved in a minimum amount of hot absolute ethanol. The resulting solution was boiled with charcoal and filtered. Anhydrous ether was added to the hot fil­ trate until clouding resulted. A white crystalline pro­ duct which was obtained on cooling was filtered and dried in a vacuum desiccator over P2 O5 . The yield was 13.8 Gm.

(81 per cent of the theoretical) of product which melted at 182* to 1850c.

The product was water soluble; an aqueous solution gave a positive test for chloride ion by using AgNOg test solution, and an oily product resulted when made alkaline with 5 per cent NaOH, indicating that the product was the 49 amide, beoause the amine itself was soluble in alkaline solution. The solid gave a positive Beilstein test for chlorine.

Percentage nitrogen for OieBgsNgO^Cl:

calculated 8 , 1 2

found 8,13

Molecular weight determination by the Mohr

titration:

calculated 344.8

found 341.7

Disubstituted amides of 2.5- dimethozybenzoio acid

Dialkvl

1, NjN-'diisobutyl- 2,5- dimethoxybenzamide

H3CO7 -

'/ \\i./

-ÔCH3

For the preparation of N,N- diisobutyl- 2,5- dimethoxybenzamlde, 2,5- dimethoxybenzoyl chloride, obtained from 0,05 mole (9,11 Gm,) of 2,5- dimethoxybenzoio acid, and 12,92 Gm, (0,1 mole) of dilsobutylamine were used. 50

The oily acid chloride was dissolved in 25 ml, of

anhydrous ether, Dilsobutylamine (0,1 mole, 12,92 Gm,) in

25 ml, of anhydrous ether was added dropwise, with constant

stirring, to the ether solution of the acid chloride, A

white precipitate resulted. The reaction mixture was

stirred at room temperature for 30 minutes to assure com­

plete reaction. The ether was evaporated, leaving a yellcw

oil in addition to the white precipitate. This mixture was washed with 25 ml, of 10 per cent hydrochloric acid to

remove any unreacted dilsobutylamine and then extracted with 25 ml, of ether. The ether extract was then shaken with 25 ml, of 5 per cent sodium bicarbonate to remove any

unreacted acid chloride and then with water, in a separa­

tory funnel. The resulting ether layer was dried over

anhydrous NagSO^, % e n the ether was evaporated, a yellow

oil weighing 12 Gm, remained. Attempts to solidify or

crystallize the oil were unsuccessful. Upon distillation

under reduced pressure, a pale yellow oily fraction, boil­

ing from 183° to 184°C, at 2 mm, Hg, was collected. This fraction weighed 10 Gm, The per cent yield of this product was 68,1 per cent.

Percentage nitrogen for C17H27NO3 :

calculated 4,80

found 4,79 51

Refraotive index:

2 0 1.5065

2. N,N- diisoamyl- 2,5- dimethoxybenzamide

\ /H. OCHj 'ch.CHvCH,^^

N,N- diisoamyl- 2,5- dimethoxybenzamide was prepared

by the reaction of 0,05 mole of 2,5- dimethoxybenzoyl

chloride, obtained from 9,11 Gm, (0.05 mole) of 2,5- dimethoxybenzoic acid, with 0,1 mole (15,73 Gm,) of

diisoamyl amine according to the procedure employed for the preparation of the diisobutyl derivative, When the ether was evaporated, following the reaction of the acid chloride and the amine, a yellow-white residue remained. This residue was washed with 25 ml. of 10 per cent hydrochloric acid to remove any unreacted amine and was then filtered.

The remaining residue was washed with 5 per cent sodium bicarbonate to remove any unreaoted acid and was dried in a vacuum desiccator over P2O5 .

The dried crude amide was dissolved in ether and the resulting solution was boiled with charcoal. After filtering, petroleum ether (30-60) was added to the 52 filtrate until clouding resulted. On cooling, white needle-like crystals settled out. After filtering and dry­ ing in a vacuum desiccator over PgOg, the white product weighed 12.5 Gm. or 85 per cent of the theoretical and melted at 61° to 62°C,

Percentage nitrogen for C19H31NO3 :

calculated 4.36

found 4.43

Diaralkvl

1, N,N- dihenzyl- 2,5- dimethoxybenzamide

H3C0 / W L ;

The starting compounds for the preparation of N,N- dibenzyl- 2,5- dimethoxybenzamide were 22.1 Gm. (0.1 mole) of dibenzylamine and 2,5- dimethoxybenzoyl chloride, which was obtained from 9.11 Gm. (0.05 mole) of 2,5- dimethoxy­ benzoio acid. The acid chloride was dissolved in 25 ml. of anhydrous ether, and the resulting ether solution was added dropwise, with constant stirring, to 25 ml. of anhydrous ether containing dibenzylamine. After the addition of the acid chloride to the amine, the reaction mixture was 53

stirred at room temperature for 30 minutes to assure com­

plete reaction. The ether was evaporated and the residue

was washed with 85 ml, of 10 per cent hydrochloric acid to

remove any unreacted amine and was then filtered. The

light yellow solid product obtained was washed with 5 per

cent sodium bicarbonate to remove any unreacted acid

chloride and then with water. The crude amide was dis­

solved in alcohol and the resulting solution was boiled

with charcoal and filtered, Water was added to the hot

filtrate until a clouding resulted. On cooling fine white

needle-like crystals separated out. After filtering and

drying in a vacuum desiccator over PgOs, the product weighed 14,5 Gm, or 80 per cent of the theoretical based on

the weight of 2,5- dimethozybenzoic acid used. The amide

melted at 91° to 9E°C.

Percentage nitrogen for OggHggNOg:

calculated 3.88

found 3.88

Miscellaneous ^ides of 2.5- dimetlioiy’benzoio acid

1, N- (2,5- dimethoxybenzoyl) pyrrolidine

For the preparation of N- (2,5- dimethoxybenzoyl)

pyrrolidine, 7,1 Gm, of pyrrolidine and 2,5- dimethoxy­

benzoyl chloride, obtained from 9,11 Gm, of 2,5- 54

H,CO-

dimethoxybenzoic aoid, were used. The aoid chloride was dissolved in 25 ml, of anhydrous ether. The resulting ether solution of the aoid chloride was added dropwise, with constant stirring, to 25 ml. of anhydrous ether con­ taining 0,1 mole (7.1 Gm,) of pyrrolidine. During the addition of the acid chloride to the amine the reaction was kept in an ice bath. When the addition of the acid chloride was completed, the reaction was stirred for 30 minutes at room temperature to assure complete reaction. The ether was evaporated and the resulting solid was washed with 25 ml. of 10 per cent hydrochloric acid to remove any unreacted pyrrolidine and was then filtered. The light yellow precipitate was then washed with 5 per cent aqueous sodium bicarbonate to remove any unreacted acid chloride, and dried over PgOg in a vacuum desiccator.

The dried product was recrystallized twice from a mixture of ether and petroleum ether (30-60), yielding 10

Gm, (85 per cent of the theoretical based on the weight of

2,5- dimethoxybenzoio acid used) of a white needle-like crystalline product which melted at 78° to 79°C., after drying in a vacuum desiccator over Pgpg. 55

Peroentage nitrogen for C13H17NO3 :

calculated 5.95

found 5,96

2, N- (2,5- dimethoxytoenzoyl) piperidine

H,CO

Piperidine (0.1 mole or 8.52 Gm,) was reacted with

2,5- dimethoxybenzoyl chloride, obtained from 9.11 Gm, (0.05 mole) of 2,5- dimethoxybenzoio aoid to give N- (2,5- dimethoxybanzoyl) piperidine according to the procedure described for the preparation of the pyrrolidine derivative.

Hew ever, when the ether was evaporated following the reac­ tion of the 2,5- dimethoxybenzoyl chloride with piperidine, a viscous oily product was obtained. This oil was washed with 25 ml, of 10 per cent hydrochloric acid to remove any unreaoted piperidine and was extracted with 25 ml. of ether.

The ether solution was shaken with 25 ml. of 5 per cent sodium bicarbonate to remove any unreacted aoid chloride and then with water, in a separatory funnel. The resulting ether layer was dried over NagSO^. Evaporation of the ether yielded 10 Gm. of a very viscous oil, which failed to solidify on standing. The oil was distilled under reduced 56

pressure glving a 5.5 Gm. (44 per cent of the theoretical)

fraction which toiled at 181® to 183®C. at 3 mm. of Hg,

This very viscous fraction solidified on standing over­ night, and this solid had a melting point of 51® to 53®0.

Peroentage nitrogen for O14H19NO3 :

calculated 5,62

found 5.61

3. 2,5- dimethoxybenzoyl urea

H.CO Y ^VCNHCWHi

\ ~ / 0 CH3

The ureide of 2,5- dimethoxybenzoio acid, 2,5- dimethoxybenzoyl urea, was prepared by the action of ammonia gas on the isocyanate of 2,5 dimethoxybenzoio aoid,

2,5- dimethoxybenzoyl isocyanate. The 2,5- dimethoxy­ benzoyl isocyanate was prepared similar to the method described by Arcus and Prydal (50), for the preparation of benzoyl isocyanate.

The acid chloride of 2,5- dimethoxybenzoio acid was prepared by the action of 8001g on 0.05 (9.11 Gm.) of the acid. The aoid chloride obtained was dissolved in 25 ml. of dry CCI4 and this solution was added dropvise to a constantly stirring slurry of 0.1 mole (14.9 Gm.) of silver 57

oyanate (AgCNO) in 25 ml. of dry COl^. After the addition

of the aoid chloride was completed, the miiture was reflured

for six hours using a reflux condenser fitted with a drying

tube to exclude moisture. The solvent was then removed by

vacuum distillation using a water pump, with dry nitrogen,

bubbling through. The isocyanate was extracted from the

crude residue with 50 ml, of anhydrous ether. The ether

solution was then filtered.

Dry ammonia gas was bubbled through the yellow

filtrate resulting in a li^t yellow colored precipitate.

The solvent was evaporated by passing a current of air over

the mixture. The resulting product was recrystallized from

ethanol using charcoal as a decolorizing agent and was

dried in a vacuum desiccator over PgOg, The dried fine

needle-like crystals weighed 7,4 Gm. (66 per cent of the

theoretical) and had a melting point of 174° to 175°C.

Percentage nitrogen for O10 B12N2O4 :

calculated 12,50

found 12,48

\ 58

The reactions involved in the preparation of

2,5- dimethoxybenzoyl urea are as follows:

M3C0 H^CO- SOC\. y \\i OH y W fC Cl

•OCH. ocv\.

2,5- dimethoxybenzoio 2,5- dimethoxybenzoyl aoid chloride

H,CO HjCo AqCNO V / \\iCCI

■ocrt. OCH,

2,5- dimethoxybenzoyl 2,5- dimethoxybenzoyl chloride oyanate

CCNO y \ \ c - . CO ocH,

2,5- dimethoxybenzoyl 2,5- dimethoxybenzoyl oyanate isooyanate

H,Co O NH> !• y \viC MCO CNHCNH,

OCH,

2,5- dimethoxybenzoyl 2,5- dimethoxybenzoyl isooyanate urea INFRARED SPECTROSCOPY

The degree of moleoular motion possessed by a molecule is dependent upon the amount of energy it possesses. If we assume we have a molecule completely devoid of energy, it has not motion. If it is given energy, say in the form of radiant energy, motion begins and it can take any one of or combination of four types of motion depending upon the amount of energy supplied to it.

Mathematical treatment may be given to these four forms of motion for very simple molecules in which the four forms are assumed to be absolute, but in more complex molecules mathematical treatment cannot be realized.

These four forms of motion are ranked as follows, according to the energy requirements (51), (1) Transla­ tional, in which the molecule moves from one point in space to another. The amount of energy required for this type of motion is in the order of a few hundred calories per mole, and in the spectral scale, radiant energy of a very low order (twenty microns in wave length) is sufficient to initiate this motion, (2) Rotational, in which the mole­ cule rotates about some central axis. Molecular motion of this type is initiated by energies in the order of one thousand calories per mole and the radiant energy falls in 59 60 the range of twenty microns, (3) Vibrational, in which atoms are displaced from their position and they oscillate back and forth or move sideways with a swinging motion within the molecule, From one thousand to about forty thousand calories per mole are required for this motion depending, of course, upon the size and the nature of the molecule. The rotational motions, involving small ener­ gies, are superimposed on the atomic displacement, giving rise to absorption bands in the near infrared region of the spectrum which extends from about two to sixteen microns,

(4) Electronic, in which an outer electron is displaced within the molecule. The only difference between ultra­ violet and visible absorption spectra is that greater energies and greater displacements are involved in ultra­ violet absorption— thirty-five thousand to seventy-one thousand calories per mole being required for absorption in the visible region of 4000 to 8000 A® and from seventy-one to several hundred thousand calories per mole in the ultra-violet region of 2000 to 4000 A°,

Infrared absorption then are concerned with mole­ cules which are capable of rotation and vibration in the infrared spectral region.

There is an additional requirement, however, A compound most be sufficiently asymmetrical to possess a dipole moment to absorb in the infrared region. Vibration 61 of two similar atoms against eaoh other, as for example nitrogen or oxygen molecules, will not result in a change in the electrical symmetry of the molecules and thus such molecules do not absorb or are inactive in the infrared spectral region.

The most significant portion of the infrared region of the spectrum for structure determination is the region from two to eight microns, for it is in this region that individual bands are more or less characteristic of specific pairs or groups of atoms. Above eight microns, the bands are due to vibrations and rotations in which all of the atoms of the molecule take part as a unit.

The spectrographs of pure compounds are so highly specific that they have become accepted as the "finger­ prints" of those compounds. Very slight traces of impurity will change the spectrograph of the compound. For example, as little as 0,3 per cent of 1,2- dibrompopropane may be qualitatively as well as quantitatively determined in 0 .1 ml. of 1,3- dibrompopropane (52),

When a compound has undergone a chemical reaction, the fingerprint changes to that characteristic of the new compound but the groups of atoms that are present in the parent compound plus those groups of atoms present in the new compound with which the parent compound was reacted will quite often show their characteristic absorption 62

bands. Absorption in any other particular band, however,

is dependent upon the group of atoms itself plus the

neighboring groups of atoms. The other structures adjacent

to a given group of atoms influence its electronic and

spatial configuration. According to Brode (53), a

homologous series tends to show differences in intensity

rather than differences in frequency although some shifting

may occur,

A further consideration is the effect of the solvent

used upon the spectrograph. The ideal situation is that of

a liquid compound in which the absorption observed is due

to the molecules of the compound only, IThen the compound

is a solid, it must be dissolved in some solvent or sus­

pended in some liquid. Solvents such as chloroform or

acetone can be used, and the most widely used suspending

liquid is Nujol, The liquid used as a solvent or suspend­

ing agent must not react with the cells, which are made of

rock salt. The bands due to the presence of the solvent of

suspending agent must be considered. Another method of

handling a solid compound is to prepare a solid suspension,

i,e., a suspension of the compound in another solid. Since

KBr does not absorb to any great extent in the infrared

region, this compound is used as a solid suspending agent.

So called ”KBr pellets” are then used in place of a solution or liquid suspension of the compound. The 63 resulting spectrograph will then be essentially the spectrograph of the compound alone.

These factors then place the determination of the structure of a new compound somewhat on an empirical basis.

There is some uncertainty present, but when the information obtainable from am infrared spectrograph is combined with other analytical data such as carbon-hydrogen or nitrogen analyses, plus the knowledge of how the compound was pre­ pared, i,0 ,, what the starting products were, etc., valid conclusions may be drawn as to the structure of the new compound.

Numerous infrared spectrographs of known compounds have been compiled, such as by Randall et al, (54), which show that certain functional groupings of atoms in a mole­ cule absorb regularly at specific wave lengths. Table I presents the absorption bands for some specific groupings of atoms in molecules from known compounds as compiled by the Department of Chemistry, The Ohio State University,

Only the absorption bands characteristic of the functional groupings that would be present in the new amides prepared and described above are presented here. 64

TABLE I

ABSORPTION BANDS FOR KNOWN GROUPS OF ATOMS AS COMPILED BY THE OHIO STATE UNIVERSITY

Functional Group Wave Length in Microns

Monosubstituted Amides 2.94 to 3.26 5.84 to 6.13 5.62 to 6.75

Disubstituted Amides 5.86 to 6.13

Amine Hydroobloride 3.62 to 4.20

Acid Carbonyl 3.03 to 3.45 5.90 to 5.95

Ethers 8.95 to 9.15

Ureido 5.81 to 5.99

Phenyl Ring 6.15 to 6.70

Aromatic C - H 3.20 to 3.35

Aliphatic C - H 3.33 to 3.71 INFRARED SPECTROGRAPHS AND THEIR INTERPRETATION

As stated previously, the infrared spectrographs of

the N- substituted amides of 2,5- dimethoxybenzoio acid

which were viscous liquid (Figures 2 to 4, and 18) were

prepared using the pure oil. The infrared spectrographs of

all the solid N- substituted amides and of 2,5- dimethoxy­

benzoio acid (Figures 5 to 6 and 19 to 23) as well as the

infrared spectrograph of 2,5- dimethoxybenzoio acid (Figure

1 ) were prepared by using potassium bromide pellets.

The infrared spectrograph of 2,5- dimethoxybenzoio

acid (Figure 1 and Table II) shows characteristic absorp­

tion bands attributed to the presence of the oarboxylio

acid carbonyl function, at 3,20 and 5.75 microns. The

absorption band at 3,20 microns should shift to the region

of 2,94 to 3,26 microns, when the caiboxylic acid function

is reacted to form an N- monosubstituted amide. The

absorption band found after the transformation is attributed

to the N - H bond. The infrared spectrographs of the mono­

substituted amides of 2,5- dimethoxybenzoio acid show this

characteristic shift of the oarboxylic acid carbonyl

absorption band to a lower wave length (Figures 2 to 17 and

Tables II to VIII), On the other hand if the oarboxylio

65 66 aoid is reaoted to form a disubstituted amide, the absorp­ tion band in this region disappears beoanse of the absence of the N - H bond. The infrared speotrographs of the com­ pounds in Figures 18 to 20 (Table IX) do not show absorp­ tion bands in the region of 2,94 to 3,26 microns. These compounds are disubstituted amides and since no N - H bond would be present, no absorption would take place. Simi­ larly, the infrared speotrographs of N- (2,5- dimethoxy­ benzoyl) piperidine and N- (2,5- dimethoxybenzoyl) pyrrolidine (Figures 21 and 22 and Table X) do not show absorption bands in the region of 2,94 to 3,26 mioi'ons.

These two compounds are also disubstituted amides in which the nitrogen atom is contained in a ring.

A second shift of the carboxylic aoid carbonyl absorption band at 5,75 microns occurs when an N- substi­ tuted amide is formed from the acid. This shift is characterized by a new absorption band at approximately

6,00 microns for monosubstituted amides and approximately

6,10 microns for disubstituted amides. All the infrared speotrographs of the N- substituted amides of 2,5- dimethoxybenzoic aoid either monosubstituted or disub­ stituted (Figures 2 to 23 and Tables II to XI) show absorption bands in this region which confirm substituted amide formation. 67

The infrared speotrographs of the N- substituted amides of 2,5- dimethoxybenzoio aoid prepared as hydro- ohloride salts of an amino funotion (Figures 5, 6 , 14, and

16 and Tables IV, VI, and VIII) show absorption bands oharaotsristio of the amine hydrochloride (3,70 to 4,10 microns),

In addition to the oharaoteristic absorption bands of monosubstituted amides, the infrared spectrograph of

8,5- dimethoxybenzoyl urea (Figure 23 and Table XI) shows absorption bands characteristic of a second carbonyl func­ tion, i,e,, at 3,20 and 5,70 microns. These absorption bands are characteristic of a ureido carbonyl. WAVE NUMBERS IN CM « WAVE NUMBERS IN CM ' 5000 4000 3000 25002000 1500 1400 1300 1200 i 100 1000 900800 700 625 100 100

80

= 60

40

20

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 1

Infrared Spectrograph of 2 , ^ Dijnethaxybenzoic Acid 69

TABLE II

ABSORPTION BANDS FOJND IN 2,$- DIMETHCfiCïBENZOIC ACID

Figure Functional Groups Wave Length in Microns

1 Garboxylic Acid Carbonyl 3.20

Carbooylic Acid Carbonyl 5.75

Aromatic Ring 6.1$

Aromatic Ring 6.6$

Ether (- OGH3 ) 7.70 WAVE NUMBERS IN CM ' W AVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 MOO 1000 900 800 700 625 100 ICO

= 60 60 =

40

20

2 3 4 5 6 7 8 9 ID 12 13 15 1614 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 2

Infrared Spectrograph of N- n- Propyl- 2 , ^ Dimethooqybenzaniide

SI o W AVE NUMBERS IN C M > WAVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900 800 700 100 100

80

= 60 60 =

^ 40 40

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 3

Infrared Spectrograph of N- n- ButyL- 2,5- Dimethoxybenzaiaide

H WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM ' 5000 4000 3000 7500 2000 1500 1400 1300 1200 1100 >000 900 800 700 625 100 [100

60 =

20

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure L

Infrared Spectrograph of N- Isoamyl- 2,5- Dimethaxybenzamide

ro 73

TABLE III

ABSORPTION BANDS FOUND IN N- ALKYL SUBSTITUTED AMIDES OF 2 , 5 - DDŒTHCKYBENZOIC ACID

Figure Functional Group Wave Length in Microns

2^ Monosubstituted Amide 2.9U

Monosubstituted Amide 6.00

Aliphatic 0 - H 3.1i$

3^ Monosubstituted Amide 2.95

Monosubstituted Amide 6.00

Aliphatic G - H 3.1|6

1^° Monosubstituted Amide 2.95

Monosubstituted Amide 6 . 0 0

Aliphatic G - H 3 4 5

a N- n- propyl- 2,5- dimethoocybenzamide • n- butyl- 2,5- dimethotybenzamide. °N- isoanyl- 2, 5 - djjnethoxybenzamide. WAVE NUMBERS IN C M * WAVE NUMBERS IN CM * 625 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900 800 700 100 100

60^

40 Ü

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure S

Infrared Spectrograph of N-V - Dime thy laminopropyl- 2 , 5 - Dimethoxybenzamide %rdrochloride WAVE NUMBERS IN C M ' WAVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900 800 700 625 100 ,100

80

60

20 ]20

2 34 5 6 7 8 9 10 11 12 13 14 15 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 6

Infrared Spectrograph of N-V - DiethyTaminoproporl^ 2,^ Dimethoxybenzamide I^ydrochloride vn 76

TABIE IV

ABSORPTION BANDS FOUND IN N- DIALKÏIAMINOALKÏL SUBSTITUTED AMIDES (F 2 , 5 - DIMETHCECYBENZAMIDE

Figure Functional Group Wave Length in Microns

Monosubstituted Amide 3 . 0 0

Monosubstituted Amide 6.05

Aliphatic G - H 3.1^6

Amine I^rdrochloride

6b Monosubstituted Amide 2.97

Monosubstituted Amide 6.03

Aliphatic G - H 3 .I18

Amine I^drochloride 1 .1 5

^N-Y - dimethylaminopropjrl- 2,5- dimethoxybenzamide hydro­ chloride • “ diethylaminopropyl- 2,5- dimethaxybenzamide hydro­ chloride • WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM ' 5000 4000 3000 25002000 1500 1400 1300 1200 1100 1000 900 800 700 625 100 100

80

60

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 7

Infrared Spectrograph of N- Qyclohexyl- 2 , ^ Dimethoaybenzamide 78

TABIE V

ABSORPTION BANDS FOUND IN N- CYCLOHEEfl^ 2,5- DIMETHCKÏBEN2AMIDE

Figure Functional Group Wave Length in Microns

7 Monosubstituted Amide 2.97

Monosubstituted Amide 6.05

Gy c lie - CH2 - 3*h6

Cyclic - - 3.58 WAVE NUMBERS IN C M ' W AVE NUMBERS IN C M ' 7 005000 4000 3000 2500 p2000 1500 1400 1300 1200 1100 1000 900 800 7005000 625 too 100

r-- 80

2 34 56 7 8 910 12 13 14 15 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 8

Infrared Spectrograph of N- 2*- Methylphengrl- 2 , ^ DimethoKybenaamide

-a VO WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM ' 5000 4000 3000 2500 2000 1400 1300 1200 1100 1000 900 800 7001500 625 100 100

60 60 =

0 i- WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 9

Infrared Spectrograph of N- 3'- Methylphenyl- 2,9- Dimethoxybenzamide

03 o WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM ' 5000 4000 3000 2500 ' 2000 1500 1400 1300 1200 1100 1000 900 800 700 625 100 100

I z<

40 40 UI

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 10

Infrared Spectrograph of N- i»’- Metbylpheiyl- 2,5- DimethoxybenzanuLde

CO PF.RCENT TRANSMITTANCE

4»-....

§z

o- !-----

ft g K

29 PERCENT TRANSMITTANCE WAVE NUMBERS IN C M • WAVE NUMBERS IN C M > 5000 4000 3000 2500 1 2000 1500 1400 1300 1200 1100 1000 900 800 700 625 lOOr ;I00

W< 1 I i - 40 Ü

20 — -— ,20

2 3 4 5 6 7 8 9 10 12 13 14 IS 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 12

Infrared Spectrograph of N- 2»,5‘- Dimethjjrlphenyl- 2,5- Dimethoxybenzamide WAVE NUMBERS IN CM ' WAVE NUMBERS IN C M ' 5000 4000 3000 25002000 1500 1400 1300 1200 1100 1000 900 800 700 625 too — 100

i 60

•40

-20

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 33

Infrared Spectrograph of N- 2*, U ‘- Dime thy Ipheiyl- Dimethoaybenzamide WAVE NUMBERS IN CM ' W AVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900 800 700 625 too 100

80

60 60 i t 40 40^

20 20

2 3 4 5 6 7 89 10 12 14 1513 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 3li

Infrared Spectrograph of N- p- Diethylaminophenjrl- 2,5— Dimethoxybenzamide hydrochloride

CO vn 86

T A B I £ V I

ABSORPTION BAl'IDS FOUND IN N- AROHATIC SUBSTITUTED AMIDES (F 2,$- DIMETHCKIBËNZQIG ACID

Figure Functional Group UKave Length in Microns

8^ Monosubstituted Amide 3*00

Monosubstituted Amide 5*95

9b Monosubstituted Amide 3.02

Monosubstituted Amide 5.95

10 ° Monosubstituted Amide 3.05

Monosubstituted Amide 5*95

u d Monosubstituted Amide 2.99

Monosubstituted Amide 5.95

12 ® Monosubstituted Amide 2*99

Monosubs titute d Amide 5.95

13 ^ Monosubstituted Amide 2.96

Monosubstituted Amide 5*95

1 ];S Monosubstituted Amide 3.05

Monosubstituted Amide 5*99

Amine Hydrochloride li*25

2 »- methylphenyl- 2 ,5 - dimethoxybenzamide* bjj- 31 - methylphenyl- 2 ,5 - dimethoxybenzamide * °N- U'- methylphezyl- 2 ,5 - dimethoxybenzamide* <%. 2 *,6'- dimethylpheqyif. 2 ,5 - dimethoxybenzamide * ®N- 2',5'- dimetbylpherylp* 2,5- dimethoxybenzamide* % - 2 ',U'- dimetfaylphenyL* 2 ,5 - dimethoxybenzamide. SN- p - diethy]aminophenyl- 2,5- dimethoxybenzamide %dro- chloride * WAVE NUMBERS IN C M * W AVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900 800 700 100 ilOO

80

fc 40 40^

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 1$

Infrared Spectrograph of N- Ben^l- 2,5- Dimethoxybenzamide Oo WAVE NUMBERS IN C M « W AVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 900 800 700 100 ilOO

60^

40

WAVE LENGTH IN MICRONS WAVE l e n g t h IN MICRONS

Figure 16

Infrared Spectrograph of N- yB - Phenylethyl^ 2,5- Dimethoxybenzamide

OO CD WAVE NUMBERS IN CM^' WAVE NUMBERS IN CM > 5000 4000 3000 2000 1500 1400 13002500 IlOO 1000 900 800 700 «5 100 100

#0 y

fc 60 60 =

5 ^ 40^

2 3 4 56 7 8 9 10 II 12 13 14 15 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 17

Infrared Spectrograph of N - Y «-* (ij^ Morpholino) propyl- 2,5— Dimethoaybenzamide hydrochloride

cx> NO 90

TABI2 VII

ABSCRPTXON BANDü FOUND IN N- ARAUffL SUBSTITUTED AMIDES CF 2,5- DIMETHOCIBENZOIC ACID

Figure Functional Group Wave length in Microns

Monosubstituted Amide 3 .0 0

Monosubstituted Amide 6.05

Aliphatic C - H 3.46

Aliphatic C - H 3.65

16b Monosubstituted Amide 2.91

Monosubstituted Amide 5.99

Aliphatic C - H 3.41

Aliphatic C - H 3.51

^N- ben^l- 2 ,5 - dimethoosybenzamide* % - /3 - phenylfithyl- 2,5- dimethoosybenzaunids. 91

TABLE VIII

ABSORPTION BANDS FOUND IN N- Y - (L- MCEPHOIINO) PROFIL- 2,5- DIMETHCECYBENZAMIDE

Figure Functional Group Wave Length in Microns

17 Monosubstituted Amide 3.00

Monosubstituted Amide 6.05

Aliphatic C - H 3.50

Amine Hydrochloride U*io

— CH2 — N — CH2 — 9.00 WAVE NUMBERS IN CM ' W a v e n u m b er s in c m -» 5000 4000 3000 2500 2000 1500 1400 1300 1200 IlOO 900 800 700 625 ICO 100

60 60fc

20

W A VE LE NG T H IN MIC RO N SWAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONSWAVE

Figure 18

Infrared %)eotrograph of N,N- Diisobutyl- 2,5- Dimethoxybenzamide % WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM * 5000 4000 2500 2000 1500 1400 1300 1200 1100 1000 9003000 800 700 100 100

80

= 60 60 =

^ 40

20

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 19

Infrared Spectrograph of N,N- Diisoaiqyl- 2,2- Dimathoxybenzamide WAVE NUMBERS IN CM ' WAVE NUMBERS (N CM ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900800 700 62S 100 100

80

60 60 =

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 20 Infrared Spectrograph of Dibenzyl- 2,3- Dimethaxybenzaioide WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 000 700 625 100 100

I 60 =

S

20

2 3 4 5 6 7 8 9 10 II 12 13 14 IS 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 21

Infrared Spectrograph of N- (2,5- Dimethoxybenzpyl) Piperidine WAVE NUMBERS IN CM ' WAVE NUMBERS IN C M ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 1100 1000 900 800 700 625 100 \r\j 100

60 =

5 «

WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 22

Infrared Spectrograph of N- (2,5- Dimethcœybenzqyl) lyrrolidine

» 97

TABLE IX

ABSORPTION BANDS FOUND IN N,N- DISUBSTITUTED AMIDES CP 2 , ^ DIMETHCEYBENZOIC ACID

Figure Functional Group Wave Length in Microns

1 8 ^ Aliphatic C - H 3.U5

Disubstituted Amide 6 .1 0

19b Aliphatic C - H 3.U5

Disubstituted Amide 6 .1 0

20 P Disubstituted Amide 6.08

®N,N- diisobutyL- 2 ,5 - dlmethojybenzaiiiide • ^,N- düsoançrl- 2 ,5 - dimethaxybenzamide. ®N,N- dibenzyl- 2,5- dimethaxybenzamide. 98 TABLE X

ABSORPTION BANDS FOUND IN N- (2,5- DDŒTHCKÏBEN20ÏL) PIPERIDINE AND N- (2,5- DIMETHOdBENZOTL) PIRROUDINE

Figure Functional Group Wave Length in Microns

21 ^ Qyclic - CHg - 3.50

Qyclic - CHg - 3.59

Disubstituted Amide 6.15

22 ^ Qyclic - OHg - 3.1*5

Cyclic - CHg - 3.50

Disubstituted Amide 6.15

*N- (2 ,5 - dimethoixybenzoyl) piperidine. (2 ,5 - dimethoxybenzcyl) pyrrolidine.

TABIE XI

ABSORPTim BANDS FOUND IN 2,5- DIMETHCKrEEMZOrL UREA

Figure Functional Group Wave Length in Microns

22 Monosubstituted Amide 3.00

Monosubstituted Amide 5.95

Ureido Garbooyl 3.2 0

Ureido Carbonyl 5.70 WAVE NUMBERS IN CM ' WAVE NUMBERS IN CM ' 5000 4000 3000 2500 2000 1500 1400 1300 1200 MOO 1000 900 800 700 625 100 100

80

60 =

<- 40^

2 3 4 5 6 7 8 9 10 14 15 16 WAVE LENGTH IN MICRONS WAVE LENGTH IN MICRONS

Figure 23

Infrared Spectrograph of 2,5- Dimethoxybenzoyl Urea OPTICAL PROPERTIBS

All the crystalline N- substituted amides of 2,5-

dimethoiybenzoic acid were observed using the pétrographie

microscope. They were all found to be anisotropic when

observed under crossed Nicols. All of these compounds

showed parallel extinction with the exception of N- p- diethylaminophenyl- 2,5- dimethoxybenzamide hydrochloride, which exhibited oblique extinction. Observation of the interference figures shewed that all of these compounds were also biaxial and optically negative.

The alpha and gamma refractive indices of these compounds are given in Table III.

100 101

TABIE XII

ALPHA AND G A M M RSFHAGTIVS INDICES ΠTHE CRYSTALLINE N- SUBSTITUTED AMIDES OF 2,^- DBJETHQKYBENZOIC ACID

Refractive Indices Conçound Alpha Gamma

N- Y - DimethylaminopropFyl- 2,5- Dianethoxy- benzamide hydrochloride 1.U61 1.605

N- V - Diethylaminopropyl- 2,$- Dimethoxy­ benzamide hydrochloride 1.178 1.610

N- C^clohexyl- 2,5- Dimethraqybenzamide 1.478 1.705

N- 2’- Methylphenyl- 2,5- Dimethaxybenzamide 1,484 1.609

N- 3'- Methylpheiyl- 2,5- Dimethaxybenzamide 1.478 1.614

N- U'- MethylfAienyl- 2,5- Dimethoxybenzamide 1.473 1.625

N- 2’,6*- Dimethylphenyl- 2,5- Dimethaxy­ benzamide 1.489 1.652 N- 2',5*- Dime thy Iphenyl- 2,5- Dimethoxy­ benzamide 1.490 1.692

N- 2*,U'- DimethyIphenyl- 2,5- Dimethoxy­ benzamide 1.478 1.713

N- p- Diethylaminopheiyl- 2,5- Dimethaxy­ benzamide hydrochloride 1.453 1.629

N- Benzyl- 2,5- Dimethoxybenzamide 1.484 1.733

N - Y - (li- Morpholino) Propyl- 2,5- Dimethajgr- benzamide hydrochloride 1.479 1.622

N,N- DiieoEunyl- 2,5- Dimethoxybenzamide 1.497 1.636

N,N- Dibenzyl- 2,5- Dimethaxybenzamide 1.473 1.699

N- (2,5- Dimethoxybenzoyl) Piperidine 1.445 1.733

N- (2,5- Dime thoxybenzoyl) Pyrrolidine 1.451 1.629

2,5- Dimethoxybenzoyl Urea ; 1*444 1.629 DISTRIBUTION STUDY

One of the physiooohemioal properties which may

determine the biologic activity of a drag is its distribu­

tion coefficient; a measure of the drug*s lipid solubility

as compared to its water solubility.

The Meyer-Overton theory of narcotic and hypnotic

activity is the classical example which correlates biologic

activity and distribution coefficient. In addition to the

correlation of hypnotic and narcotic activity to distribu­

tion coefficients, Sabalitschka and Tietz (55) correlated

the antibacterial properties of esters of p- hydroxybenzoio

with their respective distribution coefficients, Meyer-

Overt on and Sabalitschka showed that an increase of dis­

tribution coefficient (solubility in lipid/solubility in water) was accompanied by an increase in biologic activity,

A distribution study was conducted on the four

compounds which were prepared as hydrochloride salts of a

tertiary amino function; N-y - dimethylaminopropyl- 2,5-

dimethozybenzamide hydrochloride, N-y- diethylaminopropyl-

2,5- dimethoxybenzamide hydrochloride, N- p- diethylamino-

phenyl- 2,5- dimethoxybenzamide hydrochloride, and

N - y - (4- morpholino) propyl- 2,5- dimethoxybenzamide

hydrochloride. These compounds were selected because their

102 103 distribution coefficients could be readily obtained by titrating the free amino group by means of a nonagueous titration.

The free bases were obtained by dissolving the hydrochloride salts in water and making the solution strong alkaline with 10 per cent sodium hydroxide. The free base was then extracted from the cloudy aqueous solution with chloroform. The chloroform solution of the free base was then evaporated to dryness and the residue was dried in a vacuum desiccator over PgOg.

Approximately 0.01 M solutions of each free base were prepared by dissolving the base in dry chloroform.

These stock solutions were equilibrated to 25® 0. in a con­ stant temperature bath. Three 10 ml, samples of each stock solution were titrated with approximately 0.01 M perchloric acid in dioxane. One drop of 0.1 per cent methyl red in methanol was used as the indicator (56),

Samples of 25 ml, of the stock solutions were mixed with 25 ml. of the various aqueous buffer solutions in glass stoppered bottles. These mixtures were mechanically shaken in a constant temperature bath at 25® C. for one hour. After shaking, the mixtures were permitted to stand overnight in the constant temperature bath to Insure com­ plete separation of the layers end to obtain equilibrium. 104

Samples of 10 ml, were withdrawn and titrated with the same standard dioxane solution of perohlorio aoid.

Three determinations of the distribution coefficients were conducted at each pH.

The buffers of pH 1.15, 1.3, 1.45, and 1.65 were prepared with the KOI - HCl system described in the united

States Pharmacopeia, Fifteenth Revision. A sodium acetate- acetic acid system was used for the preparation of the buffers of pH 4.0, 4.55, and 4.95. Mono and dibasic sodium phosphates were used for the preparation of the buffers of pH 6.5, 7.0, 7.35, and 7.6 (57). The ionic strength of all of the buffers was approximately 0 .2 .

Table IIII gives the data and sample calculations of the distribution coefficients (K) using N - y - dimethyl­ aminopropyl- 2,5- dimethoxybenzamide.

A plot of the log K values vs. the pH values gave the curve in Figure 24. The slope of the curve was calculated by the least squares method.

The results of the distribution studies are given in Table H T and Figure 24 shows the curves obtained by plotting the log K values vs. the pH values. 105

TABLE ni l

DISTRIBUTION COEFFICIENTS (K) OBTAINED WITH N- y -DIMETHÏLAMINOEROPYL- 2,5- DIMETHQXyBINZAMIDE

pH of ml, to ml, to titrate buffer* titrate sample** stock solution** log K

6,5 4,76 8,27 1,36 0.1322

7.0 6,73 8,27 4,36 0,6398

7.35 7,60 8,27 11,34 1,0547

7,6 7.97 8,27 26.57 1,4243

♦measured at 25® G, with a Beokman Zeromatic pH Meter,

**an average of three titrations.

B CggQ^g/ ^buffer

“ ______ml. to titrate sample______ml. to titrate stock solution - ml. to titrate sample 106

Least Squares Method for Caloulatlng the Slope

Z values = pH values

Y values * log K values

X = 7.1 Y = 0,813

£Z = 28,45 i Y = 3,2510

^(Z)2 = 203,033 i T T . 23,915

(JX)Vn = 202,350 £ZiY/N = 83,123

1x2 = ^(Z)2 - (£X)2/n = 0,683

= (XY - ^X1Y/N = 0,792

b (slope) = £ iy/ ^ % 2 : 1,16

Y (calculated) = (Y - bX) - bX = -7,423 - bX

X = 6,5 Y (calculated) = 0,117

X = 7,0 T (calculated) = 0,697

X = 7,35 Y (calculated) = 1,103

X = 7,6 Y (calculated) = 1,393 107

TABLE U 7

DISTRIBUTION COEFFIOIENT3 (K)

H.CO

oc H

oaloulated pH of E* log K slope** R buffer log K** ■ 6.5 1.36 0.1323 0.117 7.0 4.36 0.6598 0.697 ^ CHj 7.35 11.34 1.0547 1.103 7.6 26.57 1.4243 1.393 1.16 6.5 2.48 0.3936 0.368 7.0 8.52 0.9302 0.954 7.35 20.41 1.3098 1.364 7.6 51.33 1.7104 1.657 1.17 4.0 1.49 0.1728 0.139

4.55 5.01 0.7002 0.779 4.95 19.62 1.2927 1.245 1.17 l.is 0.45 -0.5432 -0.327 1.30 0.77 -0.1113 -0.119 1.45 1.30 0.1141 0.068 ______1.65 2.22 0.3465 0.384 1.58

♦distribution ooefficient (OoEoig/Cbuffer)'

♦♦oaleulated by the least squares method. HjCO o H^CO 1.5

1.0

bO ° 0.5 / / / p 0.0 / /

- 0.5 1 4 pH

Figure 24 O Plot of the Log if the Distribution Coefficients ("log K) vs. pH eo S U M M m AND DISCUSSION

Sinoe the phenolic hydroxyls of gentisio acid are para to each other and would be susceptible to oxidation,

2,5- dimethoxybenzoic acid was used as the starting aoid for the synthesis of 22 N- substituted amides. The resulting N- substituted amides of this acid would possess two methoxyl groups para to each other and thus would not be suaoeptible to oxidation. These amides would be more stable than N- substituted amides of gentisio acid. In addition there is evidence to show that éthérification of phenolic hydroxyls tends to reduce the toxicity of the parent phenolic compound,

Gentisio aoid was etherified with dimethyl sulfate in alkaline solution to give 2,5- dimethoxybenzoic aoid.

The procedure used for the preparation of this aoid was a modification of the method originally employed by Mauthner

(49), Mauthner methylated gentisio aoid with dimethyl sulfate in alkaline solution, isolated the partially methylated product, end then remethylated the product to insure complete éthérification of the phenolic hydroxyls.

By eliminating the isolation of the partially methylated product and then rernethylating, the percentage yield of

109 110

2.5- dimethylbenzoio acid was increased from 70 per cent, reported by Mauthner, to 90 par cent.

The N- substituted amides of 2,5- dimethozybenzoic acid were prepared by the reaction of 2,5- dimethoxybenzoyl chloride with various amines. The acid chloride of 2,5- dimethoxybenzoio acid was prepared from 2,5- dimethoxy­ benzoic acid by reacting it with thionyl chloride (SOClg).

This reagent was found to be more suitable for the prepara­ tion of 2,5- dimethoxybenzoyl c hloride than was PClg. The reaction of 2,5- dimethoxybenzoic acid with PCI5 proved to be violent and any excess of PCI5 could not be removed.

The amines that were selected for the reaction with

2.5- dimethoxybenzoyl chloride may be classified as follows:

1. Alkylamines

In this category n- propyl amine (CgHi^NHg), n- butyl amine (C^HgNHg) and iso-amyl amine ( (OHg) gCHOHg^HgNHg) were chosen as typical amines for this class. These con­ stituted the lower molecular weight members of the alkylamine series.

In the study of salioylamide derivatives Way et al. ;

(5) demonstrated that N- alkyl substituted amides of salicylic aoid possessed analgetic activity. .- ° .

2. Dialky laminoalkylamines

Dime thyl aminopro pylamine ( ( CEg ) gNOEgCHgOEgNHg ) and diethylaminopropylamine ( (Gg^)gNOHgOHgOHgNHg) were selected Ill as amines of this type. The resulting H- dialkylaminoalkyl substituted amides of 2,5- dimethoxybenzoic aoid would be related to procainamide, which has been shown to possess some depressant action on heart muscle and also has local

procainamide anesthetic activity. In addition, these amides could be isolated as hydrochloride salts of the tertiary amino function giving a compound that would be in the cationic form,

5, Alioyolicamlnes

Cyolohexyl amine was used as a typical alicyclic- amine, giving an N- alicyolic substituted amide of 2,5- dimethoxybenzoio acid.

cyolohexyl amine

This derivative would be the first of a series of

N- alicyolic substituted amides. HE

4, Aromatio amines

As typical aromatic amines, 0 -, m-, and p- toluidines as well as 2,6- 2,5-, and 2,4- xylidines were chosen for the preparation of N- aromatic substituted

7

o- toluidine m- toluidine

p- toluidine

7 : //

2,6- zylidine . •' 2,5- xylidine

W3<:-<7 '^nh,

2,4- xylidine 113

amides of 2 ,5 - dimethoxybenzoic aoid. The resulting amides would be related to lidooatne which possesses local anesthetic activity.

lidocaine

Another aromatic amine, N,N- diethyl- p- phenylene- " diamine used for the preparation of another N- aromatic substituted amide of 2,5- dimethoxybenzoic acid. The amide

N,Nt diethyl- p- phenylenedlamine

• , 0 • obtained could be isolated as the hydrochloride salt giving an N- aromatic substituted cationic compound.

5, Aralkylamines

Benzylamine and p - phenylethyl amine were selected as typical aralkyl amines for the preparation of N- aralkyl amides of 2 ,5 - dimethoxy benzoic aoid. These amides are a continuation of the N- aromatic series. The amines gave 114

derivatives In which the phenyl ring is separated by one

carbon as is the case with benzylamine and by two carbons as is the case with phenylethyl amine.

benzylamine p - phenylethylamine

In addition many phenylalkyl amines are known to possess analgetic activity. Good examples of these com­ pounds are some of the sympathomimetic amines,

6 . Heterocyclic alkylamines

As a typical heterocyclic alkylamine, N- (3- aminopropyl) morphpline was selected for the preparation of an N- substituted amide of 2,5- dimethoxy benzoic acid.

The amide obtained in this case would also be related to

'• " "

. ^ ' v _ /

K- (3- aminopropyl) morphollne procaineamide. The morphollne radioal has found some application in local anesthetics as well as many other drug types. This amide also could be isolated as the 115 hydrochloride salt of the tertiary amino function end thus another cationic drug would be obtained.

7, Dialkylamines

Diisobutyl amine gOEOEgJ^gNH) and diisoamyl amine (OH.^lgOHOHgCHg 7gHG) were chosen as typical dialkyl amines. These constitute lower molecular weight members of a dialkylamine series.

Way et al. (5) also demonstrated that dialkyl substituted amides of salicylic aoid possessed analgetic activity.

8 . Diaralkylamines

Dibenzyl amine was selected for the preparation of a

N,N- dlaralkyl of 2,5- dimethoxybenzoic acid. This amide

y y

dibenzylamine would be the first of a series of N,E- diaralkyl sub­ stituted amides.

9. Cyolioamines

IVro cyolioamines, piperidine and pyrrolidine, were reacted with 2,5- dimethoxybenzoyl chloride to form disub* stituted amides in which the nitrogen is contained in a 116

ring. Piperidine is a basic ring structure in the morphine

and meperidine type analgetic drugs.

7 \ 5 fJH

piperidine pyrrolidine

Pyrrolidine would be a logical member of a series of

derivatives in which piperidine is one of the members,

10, Urea ° ” 0 H p Many acyl ureas (ureides) (R-C-N-O-NHg) possegs

hypnotic and sedative action. Among these hypnotic and

sédative ureides are carbromal, bromural, and phenacemide.

In addition, barbiturates and hydantoins are examples of

cyclic ureides with sedative action. Therefore, the ureide

of 2,5- dimethoxybenzoic acid was prepared.

The N- substituted amides prepared are divided into

three main categories,

1, Monosub stituted Amides

2, Disubstituted Amides

5, Miscellaneous Amides

The 22 compounds prepared are summarized in Tables X 7 , 1 7 1 , and X7II, 117 TABLE Xy

SUMMARï QF N- MONOSUBSTITÜTED AMIDES OF 2,5- DIMETHCKYBENZOIC ACID PREPARED

M.p. or Per Cent Per Cent Nitrogen Substituents B.p. (®0.) Y i e l d Calculated Found

A l k y l n- P r o p y l 161-163^ U5.U 6.27 6.31 n - Butyl®” 168-16 9^ Uo.o 5.90 5.87 Isoamyl 186-188^ 73.9 5.57 5.47

Dialky]aminoall

Y - Dimethylaminopropyl® 163-161 89.5 9.25 9.15

V - Diethylaminopropyl® 169-170 90.0 8.47 8.45

Alicyolic

Cyolohexyl 85-86 81.0 5.32 5.18

Aromatic

2'- Meth}»”Iphenyl 107-106 8k.5 5.16 5.19

3*- Methylphenyl 6 3 .5-61: .5 8 0 . 0 5.16 5.14

ii»- Methylphenyl 100-101 8I1.5 5.16 5.22

21,6'- Dimethy Iphenyl 121^125 8I1.5 4 . 9 1 5.02

2',5'- DimethyIp&enyl 117-118 81.5 . 4 . 9 1 5.00

2',b'- Dimethy Iphenyl 12U .5-126 8k.5 4 . 8 1 4.85

p- Diethylaminophenyl® 163-161:.5 75 oO 7.68 7.54

A r a l k y l

B e n z y l 9 0 .5-92 73.5 5.16 5.25

P - Phenyletlyl®' 238- 239^ 5 7.6 4 . 9 1 4.93

Hetrocyclic Allyl

-y- (li- Morpholino) propyl® 182-183 8 1 . 0 8.12 8.13

^Viscous liquid. ^At 2 m m . Hg. °HSjrdrochloride salt. 118

TABLE XVI

SlMiSARÏ OF N- DISUBSTITUTED AMIDES CF 2,5- DIMETHCKÏEENZOIG ACID PREPARED

Substituents M.p. or Per Cent Per Cent Nitrogen B.p. (OQ.) Yield Calculated Found

Dialkyl Diisobutyl^ 183 to 18a at 2 mm» Hg 68.1 a.80 a.79 Diisoamyl 6l to 62 85.1 a.36 a*a3 Diaralkyl Diben^l 91 to 92 80.1 3.88 3.88

Viscous liquid.

TABLE XVII

SUMMAHT ΠMISGELIAKEOUS AMIDES CF 2,5- DIMETHOXYBENZOIG ACID PREPARED

per Gent Per Cent Nitrogen Compound M.p. (°C) Yield Calculated Found

N- (2,5- Dimethoxybenzoyl) Piperidine 51 to 53 iia.o 5.62 5.61 N- (2,5- Dimethoxybenzoyl) lÿrrolidine 78 to 79 85.0 5.95 5.96 2,5- Dlmethoxybenzoyl Urea l?a to 175 66.0 12 .as 12.50 119

The infrared spectrographs of these oompounds were prepared (Figures 2 to 23). By correlating the infrared absorption bands found for these compounds (Tables II to

2 1 ) and the nitrogen analyses, the proposed structures of these gentisic acid derivatives were confirmed.

Four of the amides were isolated as hydrochloride salts of tertiary amino functions. The determination of the molecular weight of these compounds, employing the Mohr titration of the chloride ion in aqueous solution, gave further evidence to confirm the probable structures of these compounds. These compounds were:

1. N - y - dimethylaminopropyl- 2,5- dimethoxy-

benzamide hydrochloride,

2. N-y - diethylamlnopropyl- 2,5- dimethoxy-

benzamide hydrochloride,

3. N- p- diethylaminophenyl- 2,5- dimethoxy-

benzamide hydrochloride,

4. N - y - (4- morpholine) propyl- 2,5- dimethoxy-

benzamide hydrochloride.

The optical properties of the crystalline deriva­ tives were determined using a pétrographie microscope. In general they were found to be anisotropic, biaxial, opti­ cally negative crystals. The alpha and gamma refractive indices of these oompounds were determined (Table 211). 120

A distribution study was conducted on the four com­ pounds that were prepared as hydrochloride salts of a tertiary amino function; N-y - diethylaminopropyl- 2,5- dlmethoiybenzamide hydrochloride, N-y - diethylaminopropyl-

2,5- dimethoiybenzamide hydrochloride, N - Y - (4- morpholino) propyl- 2,5- dimethozybenzamide hydrochloride, and N- p- diethylaminophenyl- 2,5- dimethoiybenzamide hydrochloride.

Their distribution coefficients are summarized in Table

XI7. Figure 24 shows the plot of the log of the distribu­ tion coefficients (log K) vs. the pH of the various aqueous buffers employed in this study.

The results show that the most basic compound of the four compounds (as free bases) was the dimethylaminopropyl derivative whereas the diethylaminopropyl derivative was less basic. This could be expected due to the fact that the dimethyl compound offers six hydrogen that could hyperconjugate while the diethyl compound offers only four.

The sterio effect of the diethylamino compound is another factor that would contribute to the decrease in basicity.

This sterio effect may be the more important contributing factor.

By the substitution of a phenyl ring for the alkyl group between the amide nitrogen and the amino nitrogen, as in the N- p- diethylaminophenyl derivative, the basicity 121

was greatly reduced. This decrease in basicity is due to

the inductive effect of the phenyl ring.

The basicity of the morpholino alkyl derivative also was reduced when compared to the dialkylaminoalkyl

derivatives, apparently due to the inductive effect of the

ozygen in the morpholino ring. CONCLUSIONS

1, The éthérification of the phrnolio hydrozyla of

gentisic acid to give 2,5- dimethoxyhenzoic acid

was suitably conducted with dimethyl sulfate in

alkaline solution,

2, Thionyl chloride was found to be more suitable than

was PClg for the transformation of 2,5- dimethoxy-

benzoic acid to its acid chloride,

3, The general reaction for the preparation of the

above compounds, involving the reaction of 2,5-

dimethoxybenzoyl chloride and the various amines,

was found to be a suitable method for their prepara­

tion, since the percentage yields were relatively

high,

4, Although high temperatures were involved and some

decomposition was noted, purification of the non-

crystallizable products was accomplished by vacuum

distillation,

5» Molecular weights of the oompounds isolated as

hydrochloride salts of an amino function were suc­

cessfully determined by the titration of the

122 123

chloride ion in aqueous solution using standard

silver nitrate and potassium chromâte as the indi­

cator.

Ô* The infrared spectrographs prepared for all of the

IT- substituted amides of 2,5- dimethoxybenzoio acid

showed characteristic shifts of the absolution

bands attributed to the formation of N- monosubsti­

tuted amides and N,N- disubstituted amides,

7. Although these oompounds were not pharmacologically

tested or screened, it is hoped that this work may

be undertaken at a later date. Sufficient amounts

of the N- substituted amides were prepared so that

pharmacological testing or screening might be

carried out,

8 . A variety of amines were selected for the prepara­

tion of the N- substituted amides of 2,5- dimethoxy-

benzoic acid. However, only a few amines in each

general class of amines were used, so that a greater

variety of final products might be obtained. This

variation in the final products would give greater

scope to the future pharmacologic work. 124

9, It was found through a distribution study of the four

oompounds prepared as hydrochloride salts of a tertiary

amino function, that the basicity of the free bases

decreased in the following order:

a, N - y - dimethylaminopropyl- 2,5- dimethoiybenzamide

b, N - y - diethylaminopropyl- 2,5- dimethoiybenzamide

c, N - y - (4- morpholino) propyl- 2,5- dimethoiy­

benzamide

d, N- p- diethylaminophenyl- 2,5- dimethoiybenzamide.

Although the pharmacological activity of these com­

pounds has not been determined, this distribution study

may prove to be useful when correlated with the activi­

ties once they have been established, since the distri­

bution of a drug between an aqueous phase and a lipid

phase may determine the eitent as well as the type of

biologic activity. LIST OF REFERENCES

125 LIST OF REFERENCES

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2. Camelin, A,, Aoooyer, P., Pellerat, J., Lafuma, J,, and Coirault, R,, Bull. soo. Med. Hosp. Paris, 65, 826 (1949).

3. Camelin, A,, Steiger, R., Morel, M,, and Tary, A., Presse med., 58^ 889 (1950).

4. Sohaefer, L. S., Rasldioff, I. A., and Meigbow, R. S., Circulation, 2, 265 (1950),

5. Way, E. L., Takemori, A. E., Smith, Jr., G. S., Anderson, H. H., and Brodie, D. 0., J. Pharmacol. Exptl, Therap., 108. 450 (1953).

6 . Harthon, J. G, L., and Si grot h, K., Scan. J. Clin. Lab. Invest., 6^, 222 (1954).

7. Preziosi, J., Boll. Soo. Ital. Sper., 1443 (1953).

8 . Kapp, E. M,, and Coburn, A. F., J. Biol, Chem., 145. 549 (1942).

9. Clarke, N. E., Mosher, R. E., and Clarke, L. N,, Cir­ culation, 7_, 247 (1953) .

10. Rosenberg, E. S., Krevsky, D. A., and Kagan, B. M, Ann. Internal Med., 3^, 1513 (1952).

11. Batterman, R. 0., and Sommer, E, M., Proo. Soo, Exp. Biol. Med., 82, 376 (1953).

12. Ginoulhaic, E., Senenza, F.. iteinardi, L., Boll. Soo. Ital. Biol. Sper., 26, 383 (1950).

13. Meyer, E,, and Ragan, 0,, Federation Proo., 7, 173 (1948).

14. Pazos, I. A, ; Mon, farm. y. terap., 48 (1951),

15. Science News Letter, 72, 265 (Oct. 26, 1957), 126 127

16, Guerra, F., Soianoe, 103. 686 (1946).

17, Guerra, F,, J, Pharmaool, Exptl, Therap,, 87, 193 (1946), — "

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57, Miller, G, L., and Golder, R, H., Arch, Biochem. and Biophys., 420 (1950). AUTOBIOGRAPHY

I, Allen I, Dines, was b o m in Pittsburgh, Pennsyl­ vania, December 16, 1929, I received my secondary educa­ tion in the Pittsburgh public school system. The Univer­ sity of Pittsburgh, School of Pharmacy, granted me the

Bachelor of Science in Pharmacy degree in 1951, In 1953, the Ohio State University granted me the Master of Soienoe degree. My field of specialty was pharmaceutical chemistry.

During my Master*s program, I held a graduate assistantship from 1951 to 1952, the Upjohn Fellowship for pharmaceutical chemistry from 1952 to 1953, and completed my Master’s program, holding an American Foundation for Pharmaceutical

Education Fellowship. I continued my graduate education at the Ohio State University leading to the degree Doctor of

Philosophy as a Fellow of the American Foundation for

Pharmaceutical Education, pharmaceutical chemistry was my field of specialization.

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