THE INTERMOLECULAR COMBINATION OF ANTIMALARIAL AND ANTISEPTIC GROUPS

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n^2a,ii5s Hundert, Murray Barnard, 1919The intermolecular combination of antimalarial and antiseptic groups. New York, 19^4-9• 102 typewritten leaves, tables, diagrs. 29cm. Thesis (Ph.D.) - New York Univer­ sity, Graduate School, 1950. Bibliography: p.99-102.

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LIBRARY OP YORK UNIVERSITY OHIVERSITY HEIGHTS

■nr

THE INTERMOLECUIAR COMBINATION 07 ANTIMA.IARIAL AND ANTISEPTIC GROUPS - by -

^

MURRAY B.^HUNDERT

•October

1949

t ajUnMiTvT'u.A. t I c\

A dissertation in the Department of Chemistry submitted in partial fulfillment of the require­ ments for the degree of Dootor of Philosophy at New York University.

TABLE OF CONTENTS Page Scope of Problem

1

Introduction

3

Historical

5

Theoretical

26

General Methods of Acridine Synthesis

27

Preparation of Dialkylaminoalkylamines

48

Preparation of Antimalarials

53

Present Investigation

54

Flow Sheets

60

Physiology

65

Experimental Antimalarials from 1,1,3,3-tetramethyl-butyl phenol and o-bromobenzoio acid or o-chlorbanzoie acid

72

Antimalarials from 1,1,3,3-tetramethyl-butyl phenol and 2,4 diohlorbenzoio acid

88

Summary

98

Bibliography

99

7 0 \ Q 'I'l

To C. H.

and

R. H.

The author wishes to express his sinoere appreciation to Professor J. B. Niederl for his suggestion of the problem, his assistance generously offered, and the privilege of working with him.

-

1-

Since the 1,1,3,3-tetramethylbutyl group has shown marked bactericidal properties, it was believed that the combination of this grouping with structures known to be plasmodicidal would result in superior antimalarial compounds.

It was decided to use 1,1,3,3-tetramethylbutyl

phenol ("di/sobutyl phenol'*) as the starting material, and through the anisidino derivative, prepare both 1-(1,1,3,3tetramethyl)-butyl-4-methoxy-9-chloroacridine and 1-(1,1,3,3tetramethyl)-but yl-4-methoxy-6,9-di chloroacri dine.

The

two compounds in turn were to be condensed with dialkylaminoalkylamines which were known to enhance the antiplasmodial characteristics of the two basic compounds.

In this manner,

the dihydrochlorides of 1-(1,1,3,3-tetramethyl)-butyl-4methoxy-6-chloro-9- (3-diethylaminopropylamino)-acr.idine, 1-(1,1,3,3-tetramethyl)-butyl-4-methoxy-6-chloro-9-(5-diethylamino-2-pentylamino)-acridine, 1-(1,1,3,3-tetramethyl)-butyl4-methoxy-9-(3-diethylaminopropylamino)-acridine, and 1-(1,1,3,3-tetramethyl)-butyl-4-methoxy-9-(5-diethylamino-2pentylamino)-acridine were prepared.

Although a chlorine

atom in the 6-position was known to increase the effective­ ness of antimalarials of this type, products with and without this chlorine atom were prepared in order to better evaluate

-

2-

the contribution of the 1,1,3,3-tetramethylbutyl group. Incidental to the main thesis , a number of bactericidal compounds were also synthesized.

I N T R O D U C T I O N

-

3-

To many, the word "Malaria" implies something distant - a "jungle fever", a "tropical disease" contracted, for the most part, by semi-nude natives and treated with quinine.

The facts are that malaria attacks

a few hundred million people and kills as many as three million each year, not only in the tropics, but on every continent in the world.

And in spite of the focussing

of a tremendous concerted research effort on discovering means for controlling and eradicating the disease, man knows of no drug which is at the same time sufficiently non-toxic and prophylactic to prevent infection by all of the plasmodia parasites. A variety of factors contribute to the complexity of the problem.

For one, human malaria is known in a

number of forms, and the plasmodia responsible for the infection pass through an intricate life cycle undergoing changes not only in form but also in mode of reproduction and requiring both man and the anopholes mosquito to complete their life pattern.

For another, it has not been

possible to transmit a strain of human malaria to an ex­ perimental animal,and trial tests, therefore, must be conducted on animal hosts infected with a protozoa strain

-

4-

which differs from the one responsible for human malaria. And, in addition, the results obtained with animals are not always easily translated in terms of human chemotherapy. However, the difficult problems notwithstanding, science has made many, many notable contributions to the control of the disease, and is continuing a determined effort to improve m a n ’s defenses against the parasite re­ sponsible.

This paper reports an attempt to find a new

set of antimalarials.

H I S T O R I C A L

-

5-

Malaria - The Disease Although the cause of the infection was not known, it was long recognized that man was subjected to a group of infections which caused alternating paroxysms of fever and chills.

In 1753, Dr. Torti classified the

various fevers, and since, at the time, it was believed that bad air, (especially that coming from swamps) was responsible for the ailment, he coined the word "Malaria" ["mal" (bad) and "aria" (air)] to describe the disease. In 1898, Ross proved that it was the anopheles mosquito which was

responsible for the spread of malaria The disease itself, and

(1).

the plasmodia parasite

responsible, progress through an interesting and peculiar course.

When man is infected by

the mosquito, the

plasmodia

(called trophozoites atthis stage) find their

way into the blood stream and attack the red blood corpuscles. These parasites, at this point, divide and multiply asexually and slowly break down the red blood cells to which they are attached.

The bursting of the cells liberates

the parasite in a new form (merozoites) which again attack blood cells and again become trophozoites.

This repeated

-

6-

multiplication of the parasite and the destruction of the blood cells may continue until several cycles have been completed.

After the host builds up a sufficient

immunity, or medication causes conditions unsatisfactory for growth, some of the plasmodia evolve into male and female parasites incapable of further division or repro­ duction while in man, though still capable of destroying red blood cells.

However, since multiplication is

arrested, nature may control the disease by attacking and decreasing the level of the parasites below the minimum necessary for the disease symptoms to appear.

At this

stage, man becomes a carrier for the anopheles mosquito. To complete the cycle, the mosquito, in the process of feeding on a person having the sexual form of the parasite in his blood stream, acquires the malaria protozoa.

In the mosquito, the parasites are oapable

of sexual reproduction and give rise to sporozoites which, on reentering m a n ’s blood stream, are capable of again repeating the entire cycle. The production of fever in a patient appears to coincide with the periodic rupture of the blood cells which liberate foreign matter into the blood stream.

-

7-

Malaria and Antlmalaria3. Drugs The first recorded use of cinchona bark in combatting malaria fever has been traced back to 1630 in Ecuador (1).

Some time later, it was used in treating

the Countess Anna del Chinchon, from whom it may have derived its name.

The discovery of the use of the

cinchona bark, and later quinine, in malaria during the seventeenth century marks the beginning of m a n ’s fight against this disease.

Since quinine possesses many

clinical disadvantages, only alleviates the symptoms with­ out curing the infection (1 ), and world production of the compound can hardly satisfy demand, many investigators have sought for a synthetic compound which would be thera­ peutically more useful.

By 1930, after preparing

thousands of compounds, two drugs, "Plasmochin" and ’’Atebrine”, were found to be highly active and were offered for malariatherapy (1 ). In the five year period 1941-1946, additional potential antimalarials were prepared and tested, with the result that about seventy classes of organio compounds

-

8-

were found to possess some antimalarial action (2).

The

acridines are one of the most promising of the seventyclasses, and "Atebrine" (quinacrine), 2-methoxy-6-chloro-9(1-methyl-4-diethylaminobutylamino)-acridine-dihydrochloride/ is one of the most widely known and used synthetic anti­ malarials . The use of synthetic compounds in malariotherapy probably had its inception with Ehrlich's discovery that methylene blue bad an affinity for the malaria parasite in vitrio (3 ), and was in fact, for a time, used in conjunction with quinine.

The compound, however, really had little

or no effect on the plasmodia in vivo.

Schulemann,

Mietzsch, and Wingler (4) improved the antimalarial properties of the dye by modifying the methylene blue nucleus with dialkylaminoalkyl groups.

cHh

oH b

Methylene Blue

-

99-

rj; /

Antimalarial Compound

The acridines, also, were introduced into chemotherapy by Ehrlich.

Although acridine itself

shows a low bacteriostatic activity, many of the amino derivatives are effective germicides and are presently employed in this connection, while "Rivanol", 2 ethoxy, 6,9 diamino acridine, might be termed the first synthetic antimalarial which showed promise (3)»

Of the mono amino-

acrid ines, the 9-amino derivative is the most effective showiog the highest general bacteriostatic activity (5 ), although none are effective antimalarials.

The table

below compares the bacteriostatic activity of a number of mono amino acridines.

-

10-

Bacteriostatic Activity of Aminoacridines Activity (in powers of 2 )________

Compound Acridine

l e2

4-Amino Acridine

0.8

2-Amino Acridine

1.6

1-Amino A.cridine

1.8

3-Amino Acridine

4.2

9-Amino Acridine

4.6

What might be considered to be a combination of the 9-amino acridine and the alkylamino groupings of the type found to be effective in enhancing the antiplasmodial activity of methylene blue finally resulted in the prepara­ tion of "Atebrine" by Maus and Mietzsch.(£y 3i) c

-cH-cHvtH v'tHvyV^ H3-

T^ v

n H-

A tc . b rin e

C.I

Supposedly over 12,000 compounds were prepared and screened before "Atebrine" was developed0

-

11-

Slnce its discovery, many analogues and variations of "Atebrine" have been prepared and evaluated.

Coincident

with these investigations, some attempt was made to evaluate struoture with aotivity with limited sucoess.

Unfortunately,

the difficulties involved in evaluating the efficiency of a drug coupled with the fact that a drug may be especially useful in neutralizing one particular strain of plasmodia or attacking only one phase in the life oyole of the parasite did not permit this study to be as ezaot as desired* The following tables summarize some of the more important oonolusions possible regarding the correlation between struoture and activity based upon ollnioal tests on animals and humans with the drugs involved (7 , 8 , 12, 54)* Since the data available from different sources are not necessarily expressed in identical terms, only the groupings appearing in any one table may be compared with one another.

The efficiency of eaoh drug is expressed

either as the ratio of the maximum tolerated dose to the minimum effeotive dose (MTD/UED) or the number of grams of quinine equivalent to one gram of the drug under test (QE). In both instances, of course, the higher the numerical value, the greater the efficiency of the drug*

-

12-

For the compound:

vHf)y

y-r

Y= X= ZR--

CH 3 O Cl H (CH2 )n

The following clinical results were obtained:

TABLE I V/hen n=

Maximum tolerated dose (M T D ) Minimum effective dose (MED)

2

8

3

15

k

20

5

6

6

5

13-

j.,..

... . . . . . . . f ... .

j

t

2. A / v y* b t Y

3 o

, t { . . . . j , , r. ; } . . . . { . . . . . . 1 , ■- , |

y c d r I cn

^ .rr

s' i

j

.

{. .

f

C

b e Aw e e n

r t i t r oef* r> s

-

14-

From Table I it is readily apparent that maximum efficiency increases with increasing chain length until there are four carbon atoms between both nitrogens in the side chain.

Increasing the number of carbon atoms beyond

this point has a dystherapeutic effect on the compound. The curve shown in Graph I demonstrates these results more forcefully. In Table II are listed some clinical results obtained with compounds having the same general structure as the compounds above, but which again differ with regard to side chain.

Here, however, the effect of branching or

introducing a hydroxyl group into the side chain is evaluated.

From the data available, it may be seen that

the straight chain enhances the activity of the compound.

-

15-

TABLE II

’■There:-

Y * GH^O X - Cl Z = II

LTD MSD

and R -CH,

-CII2

-CH2

-CH2-

20

-CH|

-CH2

-CH2

-CH2-

15

-CH 2

-CH

-GH_ 2

15

■GH

-CH2

-CH2-

6.6

-CH

-CK2-

CH.,

2

cn3

-CH2

CH^

-

16-

Table III (below) compares tbe contribution to activity made by the chlorine atom, the cyanide and nitro groups when placed in the same position in a compound showing positive activity.

Here, again, the basic compound

shown on page 1£l was used.

The physiological tests show

that the chlorine atom and the cyanide group are far superior tobe. +11 to the nitro group, and the chlorine atom the most effective, A

-A

in increasing activity.

TABLE III V/hen:-

Y - CH^O R - (CH2 )3 Z -H and X =

MTD MED

Cl

15

CN

10

N °2

2.5

-

17-

Using the same basio compound for study and the nitro group and chlorine atom which were found to increase aotivity, the effect of position of these groups on activity was also ascertained.

As may be seen by comparing Tables

III and IV, the transfer of the nitro group to an adjacent carbon atom has little effect on the drug, while the same ohange made with the chlorine atom markedly influences the efficiency of the oompound.

Limited though the data may be,

the significant ohange in activity with transfer of the chlorine atom just one carbon atom away serves to emphasize the importance of both the modifying group and its position on the acridine nucleus.

TABLE IV If: Y = CH30 X =H B ^ (CH2 )3 MTD and Z no2

Cl

2.5 2

-

18-

As regards the remaining substituent (Y) Indicated In the formula plotured below, Table Y lists the effect of the methoxy, ethoxy, and methyl groups on activity. Of the three, the methoxy is obviously the most desirable modifying group showing a MTD/MED value at least double that obtained with the other two substituents.

TABLE V When: X= Cl h » ( o h 2 )3 Z= H and Y CH^O

MTD men 15

c ^ o

7.5

CH^

6

-

19-

The following results were obtained more recently (17), mainly by American investigators: For the compound: Where 1 - CH^O

X- Cl A/H

■v^l

1

TABLE VI

Rl'-( c h 2 )2-

■(gV

2-

R.,=

Ro-'

H

H

quinine Equivalent (QE)

0.1

1.5

C2H 5

°2H 5

CHo i j -c h -c h 2-

C2h 5

C2H 5

-(c h 2 )3-

H

H

somewhat active

H

CH..

somewhat active

- (CH2 )3-

1.5

-

20-

TABLE VI (Cont'd) Quinine

Rr'

R2'

-(ch2)3-

g 2h 5

•{CH2 )3-

Ry

Equivalent (Q^)

g2h 5

3.0

CH3

CH(CH3)2

2.0

■(ch2)3-

ch3

(ch2 )2ch3

3.0

(CH2 )4-

CH3

CH3

2.0

(ch2 )4-

g2h 5

c2h 5

3.5

?h 3 -CH-(CH2 )3-

CH3

C2H 5

4.0

c2h

c2h 5

6.0

GH3 -ch-(ch2 )3-

-(CH2 )5-

C2H 5

C2H 5

2.0

CHo I J •CH2-C-CH2CH3

CH3

CH3

1.5

-

21-

Alt hough there is some variation in the numeric value of the ’’quinine Equivalent” shown in Table VI above, most of the side chains are approximately equivalent in activity, since a difference of only two units is considered insignificant (7).

However, those side chains containing

four linear carbon atoms between the nitrogens (neglecting substituents) and two ethyl groups on the tertiary nitrogen atom are obviously the most effective in increasing the antimalarial activity of the parent compound.

These results are

consistent with those presented earlier. The effect of varying substituents on the four carbon chain between the two nitrogens is shown in Table VII, below.

It is readily apparent that, while a substituent on

one of these carbon atoms may be desirable, the character of the substituent is of little importance.

Thus, from the data

presented in Table VII, a phenyl group on the four carbon chain is as effective as a hexyl or methyl group.

It must be re­

membered, however, that the results presented here represent those obtained with only one test.

Further evaluation of com­

pounds of this type against a number of malaria parasites in different animals has shown a methyl group as a substituent in the four carbon ohain (the second side chain in Table VII) to be the most activating.

-

22-

TABLE VII ’.There

Y = CH3O X r Cl R 2'R3' C2h 5 quinine Equivalent

and Rj-(GH2) -

"(QE) 2.5

?H 3 -c h -(c h 2 )3-

-OH-(CH2 )3-

2.0

(CH2)5CH3 -ch-(cha )3-

3.0

I -CH2-CH-(CH2 )2-

3.0

-

23 -

TABLE VII (Cont'd.)

and R ^

quinine Equivalent (QE)

- c h -(c h 2 )3-

3.0

V I CH,

-c h -(c h 2)3-

1.5

- ( z y

3.o

Inasmuch as the major portion of the research work carried out on synthetic antimalarials of the "Atebrine" type was directed towards side chain variation, little informa­ tion is available as regards nuclear substitution.

The data

-

24-

in Table VIII (below), together with that already presented in Tables III, IV and V do not offer much in the way of correlation between nuclear substitution and activity.

The

presence of a chlorine atom in the "X" position, however, appears to be an important factor in increasing the efficiency of a drug, (see Table VIII).

TABLE VIII When:Side Chain

-NH - CH - (CH2 )^ - ( C g H ^ g CH3 and QE

Y-

X-

Test I

Test II

H

Cl

3.0

4.0

H

H

1.5

1.0

Cl

Cl

OCH^

H

OCH^

Cl

6-diamino aoridine) may be prepared using an aliphatic aldehyde (17, 18, 56).

Z

+ c//x o HHx-'ss

v

y

-

30-

Ring closure may be effected with either hydrogen chloride or zinc chloride with the elimination of ammonia.

The use of concentrated sulfurie acid at

180-200°C. will also hydrolyze the free amino groups (18).

/

i’O-j.oo'c

■r

In some cases it is possible to prepare a number of unsymmetrioal doriratires by displacing one of the amines present in the symmetrical diphenyl product with another amine or certain hydroxy oompeunds, (18, 19).

cv i

c/4,

^O'c A'H.

k

fJ#L ^

*> c//

v v tr \ ^ H

-°oti

-

o 11

vo,

Ato1

^

V / v V U

39

The scope of this type of reaotion was vastly extended by a number of Investigators*

Ullman and Goldberg

(29, 30) found that activation of the chloro group was not necessary if anhydrous potassium carbonate and oopper powder were used*

AAA

AAA

vUU

This reaction has been used most often in the preparation of baoteriostatio acridines (66, 67, 68). Other investigators have condensed aryl ketones and aldehydes with aromatic amines (31* 67) and halogenated benzenes with o-amino ketones (32) and aldehydes (67) to form various acridines and aoridones.

c" '

' „k. a

‘ */.■

/V // M

i"'

Fairly pure anthranllllc acids are possible with this reaotlon.

(7) From anthranillio esters and a Grignard compound (36 , 37):

-

43-

(8) By hydrolysis of N-phenyl isatin (38 ,64 ):

c vuH

(9) From some ortho nitro derivatives (39* 40, 41, 65):

-

44-

Through a similar Intermediate, merely heating o>nitro-diphenylmethane will give a 35# yield of acridone.

c/A ->

fro.

/vlioH

ft) I.

■

.

vX y /V-

(3S-/. f.'U)

(10) By reduotion of some suitable nitro compounds: (a)

Reduction of o,o -dinitro diphenyl

derivatives results in either aoridines or aoridones (42, 43, 44, 45, 60):

-

45-

) ii 1 ) w V '

vfYjfV N\ / \ ,/x -x

"

II

c>

(b)

Ortho nitrobenzyl chloride can

reaot with either beta naphthol or naphthylamine under reducing conditions to giro an aoridine in one step (46).

-

(c)

46-

Recently, Waterman and Vivian (47)

claimed the discovery of a new reaction wherein acridine is prepared from 2-nitrobenzophenone in the presence of . o 20# excess of ferrous oxylate dihydrate at about 220 C.

AAA

-

(11)

47-

By oxidation or "hot tube" reactions:

The unsubstituted aoridlne can be prepared by oxidation of o-aminodlphenylmethane with lead oxide (48), while the corresponding aoridone is formed by the oxidation of o-aminobenzophenone (49)•

Pio

Acridine may also be obtained by passing benzylaniline (50) or o-tolylanlline (51) through a hot tube*

-

B.

48-

Preparation of Dlalkylamino Alkylamlne Side Chains The following methods were utilized to synthesize

most of the side ohains used In the preparation and evalua­ tion of experimental synthetic antimalarials. (1) By the Gabriel synthesis (69, 70, 71, 72):

0

N -+-rXLj H ^ &L, i Z.O - / 3 o 'H-

A ri •

49-

(2) From aorylonitrile (69, 71, 73, 74) h- *• n N H

/V A

I

d

n

^a

J

r>\ t y

/3

i> C

A,

Si^lhy or A^jfcrr^i '> < y , / u

j

I^Jnty //.(/ttI I /a. i'V. >0 . / jfjo

L

/.

0r ^ ‘^

y *

A

(3)

A///

a

From amines and formaldehyde (71, 73):

A’. K,

, ,

4 1

A /;,

j

vi'

An ^

^

yv - c ^

_ c

^

- < /yt -