May 5 , 195s
BENZIhlIDAZOLES AS SPECIFIC INHIBITORS OF VITAMIN
[CONTRIBUTIOX FROM
Biz
IN
BACTERIAL MUTANTS
2165
DEPARTMENTS OF PHYSIOLOGY AND PEDIATRICS OF THE SCHOOL OF MEDICINE A N D THE DEPARTMENT O F CHEMISTRY O F THE UNIVERSITY OF PENNSYLVANIA]
THE
Benzimidazoles as Specific Inhibitors of Vitamin B1, or Thymine in Bacterial Mutants’’2 BY D. B. MCNAIRSCOTT, M. L. ROGERSAND C. ROSE RECEIVED JUNE 17, 1957 Substituted benzimidazoles have been tested on six microbial systems to determine the specificity of their inhibition of growth. SH in the 4-position produced a compound with specific inhibition of the vitamin BIZrequiring mutant and this inhibition was reversed by excess vitamin BIZ. Compounds with -NO2 in the 4-position and to a less extent in the 6-position were also inhibitory to the vitamin B12mutant and the inhibition was reversed by methionine. Alkyl or chloride substitution on the 5- and/or 6-positions produced competitive inhibitors of thymine. Compounds with two or three alkyl groups on positions 2, 5 and 6 especially were inhibitory to L.casei, the growth of which was ITmited by folic acid.
Results
Introduction
The importance of both the substituent and its Vitamin Blz has been shown to be essential for position on the benzimidazole molecule is clearly growth of many organisms, including man. Thereevident from consideration of the results obtained fore it seems possible that a specific inhibitor of vitamin BIZmight have a carcinostatic effect. 5,6- with the compounds in the different mutant systems. Dimethylbenzimidazole is one component of the The compounds which were most inhibitory to, vitamin Blz molecule. Benzimidazoles with other and reversed by, vitamin B12 are listed in Table 11, substituents might be expected to act as analogs of which expresses the inhibition in terms of the conthe vitamin BIZbenzimidazole. Hoover and Day3 centration of drug producing 50% inhibition of have synthesized a series of derivatives of benzi- growth as compared with the controls, a t the levels midazole with substituent groups, amino, nitro, metabolite indicated. chloro, mercapto and methyl mercapto, mainly on of Ten of these benzimidazoles acted as specific inthe 4- and 6-positions. These compounds and of the vitamin Blz requiring mutant, in some of a series of benzimidazoles found by Tamm, hibitors that the was reversed by increased et aLJ4to be inhibitory to the development of certain amounts of inhibition vitamin Blz or methionine. viruses, have been investigated to determine the The nitro and the mercapto groups in position 4 inhibition of growth of six microbial systems. or 6 were in competition with the vitamin B I Z . Methods and Materials.-The 49 substituted benzimida- Methionine was most effective in counteracting the zole compounds tested in this study are listed in Table I, inhibition caused by the nitro group in the 4-posiwhich also includes the source from which they were protion (Fig. l), was as effective as increased vitamin cured and the molecular weights. The organisms used were the vitamin BIZor methionine BlZagainst inhibition caused by the nitro group in requiring E. coli 113-3 or B68, purine;less E. coli B96, 1000 h thymine-less E. coli 15T- and L. casez ATCC7469 with I
I
either limiting folic acid or limiting riboflavin. The media, procedures for culture and for assay were adaptations of methods described previously.5 Results are expressed in terms of per cent. inhibition of growth compared with the control, as determined by optical density readings on the Klett-Summerson photoelectric colorimeter or by change in pH of the cultures. Screening of compounds was accomplished by incubation of non-aerated cultures containing three levels of the drug (0.01, 9.1 and 1.0 mg./ml.) and a concentration of the metabolite which permitted the control culture to attain half of the maximum growth. Where inhibition was noted, further tests were set up with increased concentration of metabolite and appropriate concentrations of the drug to determine whether the inhibition could be overcome and thus whether the drug was acting as a specific competitor of the metabolite. Studies of the effect of the compounds on the growth, cell division and synthesis of nucleic acids in cultures of the wild type E. coli B will be reported in a subsequent communication.
(1) Presented at the American Chemical Society meeting at Miami, Fla.,on April 9 , 1957. (2) This investigation was supported b y a grant (PHS C-2189) from the National Institutes of Health, Public Health Service, (3) J. Hoover and A. Day, THIS JOURNAL, 76, 4144 (1954); 77, 4324, 5652 (1955). ( 4 ) I . Tamm, K . Folkers, C. H . Shunk, D . Hey1 and F . L. Horsfall, Ex$. M e d . , 98, 245 (1953); I . Tamm, K . Folkers and C. H. Shunk, J. Bad., 72, 54, 59 (1956); I. Tamm, i b i d . , 72, 42 (1956). ( 5 ) J. S. Chiao and W. H . Peterson, Applied Microbiology, 1, No. 1 , 4 2 (1953); J. S. Gots and E. C. Chu, J . B a d . , 64, No. 4, 537 (1952); L. M. Flynn, V. B . Williams, L. O’D. Boyd and A. G. Hogan, A n a l . Chem., 23, No. 1, 180 (1951); P. Gydrgy, “Vitamin Methods,” Vol. 1, Academic Press, Inc., New York, N. Y.,p. 334.
L’ 1
0.02 OB2 VITAMIN B I Z mpg./ml.
2.0 METHIONINE
IOpg/ml. Fig. 1.-Reversal of 4-nitrobenzimidazole inhibition of growth of E. coli 113-3 by vitamin Blz or methionine Cultures of E. coli 113-3 were incubated in 5 ml. of medium for 24 hr. without aeration. They contained concentrations of compounds and vitamin BIZ or methionine as indicated, Growth was determined as turbidity with 660 m p filter.
D. B. MCNAIRSCOTT, M. L. ROGERS, .4ND
2166
c. ROSE
Vol. 80
TABLE I EENZIMIDAZOLES Code no.
1 2 3 4 5 6 7 16 17 18 19 20 25
26 28 30 33 38 42 43 44 48 54 55 56 57 or 57B
Position of substitlieti 2 I
6
n
.........
....
...
c
Iso-C~H, n-CaH, -CHrCH=CHCIIz C!zHa CH3
... ...
118
.........
. . . .... ...
l(i0
...
1' €'
, .. ....
,HZ0
160 190 116
. . .
...
.... ....
...
1
.........
......... ......... .......
.... ...
CH, CH1 (3133 CH3
...
c1
.
,
...
...
....
,
..
...
CH,
.... C1
...
...
....
...
OCHI
....
NHI
....
... .2HC1
....
N H Z
...
,2HC1
N H Z
...
.2€IC1 ... ...
NIX2 ... ... ..,
....
... NO,
CHI CHI
c1
...
....
...
...
c1
...
...
., , ,, ..
....
....
58
..
...
63 66 67 68 73
......... .........
.........
...
....
CHs
-CH&!H(
Salt
....
, ,
90 91 99 136 147 148 190 163 164 172 174 213 214 215 216 217 Source: C
...
...
6
......... .... .... .........
. .
.... ....
....
......... ......... .........
...
... ...
.........
88
, . .
CH, C4H9
...... ...... ...... ......... .........
... , . ,
,
.........
.........
. .
....
.........
8-D-Ribofuranosyl ~~-D-G~ucos~I
...
CHr
c1 c1 IT02 c1 KO2
SH c1
... ...
SH SH NHI
...
NH*
...
SOzOH
XHI
NH2)COOH
.... .... . I . .
.... .... .... ....
......... .........
....
.........
....
......... .........
.... ....
8-D-Ribofuranosyl
....
...
c1
....
NOn h-1
...
SOzNHi
...
c1
...
SH c1
NO1 NHa SCH, SCHt NOz
c1
.........
NHt ...
...
SOtNHz NO2 N H 2
...
c1
.... C
... ...
... ... ... .HCl .HCl .HCl.H2O ... ... ,2HC1
... ... ... .2HC1 ...
... .HC1 ... .2HCl.'/pC6H6
... ...
-CHzCHzCOOH ... ... ... Diethyl-N-4-(2-benzimidazolyl)-ethylaminobenzoyl glutamate HCl chloride N-( l-Benzyl-2-benzimidazolylmethyl)-pyridinium N-(1-Benzyl-2-benzimidazolylmethyl)-3-carboxamidopyridinium-chloride 1,4-Bis-(5-methyl-2-benzimidazolyl)-butane Diethyl-N-4-(5-chloro-2-benzimidazolyl)-isopropylaminobenzoyl glutamate N-(cu-2-BenzimidazolyIethyl)-pyridiniumchloride N-(2-Benzimidazolylmethyl)-3-carboxamidopyridinium chloride Diethyl, ~-hydroxyethyl,-2-benzimidazolylmethylammonium chloride = Commercial. P = Chemistry Department, University of Pennsylvania. E = Eastman.
the 6-position, and was less effective than vitamin B I when ~ the growth was inhibited by the 4-mercapto substitution (Fig. 2). Methylation of the mercapto group decreased it inhibitory capacity. While inhibition by 6-nitrobenzimidazole was reversed by increased vitamin B12, the slope of the reversal lines was less than with the 4-nitro compounds. Substitution of chlorine on the 4-position on the 6-nitrobenzimidazole changed the pattern of inhibition, decreasing the ability of vitamin BIZto reverse the inhibition as compared with the 6nitro compound (Fig. 3). The substituent on posi-
hIo1 w t 9,iiirce"
132 132 146 146 188
P P P
E M M 31 bI
318 296 P 148 M 206 P 221 P 206 P 178 P 163 P 160 M 160 i M 187 M 198 P 198 P 20-1 P 232 P 222 P 205 P P 195 238 P P 213 153 P 242 P 235 P 187 P 175 P 249 P 209 P 291 P 163 P 354 M P 190 466 P 336 P 379 P 318 P 515 P 260 P 289 P 284 P M = Mcrck.
tion 4 would seem to have more influence on the pattern of activity than the substituent on position 6. Inhibitions by 4,6-dichlorobenzimidazole and 5,6-dimethylbenzimidazolewere partially relieved by vitamin B1, or methionine. The inhibition of growth of E. coli 113-3 due to the other alkyl substituted benzimidazoles was not reversible by vitamin Bl2 or methionine. However, methionine was partially effective in reversing these same conipounds in the purine-less B96 system. Table 111 includes compounds which inhibited
Riay 5 , 1958
BENZIMIDAZOLES AS SPECIFIC INHIBITORS OF VITAMINBlz
IOOO\
0 c
BACTERIAL MUTANTS 2167
INHIBITION OF GROWTH OF
1
2
IN
4CI 6N01 B E N Z I M I D A Z O L E
1
E
Coil
6N0,
113-3
BENZIMIDAZOLE
i
:
90 O' 100' 0 -D-D
1
3 MC
2
02
20
o-o-D-0
01
02
I
20
1 20
m l VIT B 1 2 IN 10 ml
Fig 3.-Patterns of growth of E coli 113-3 inhibited by 4-chloro- 6-nitrobenzimidazole and 6-nitrobenziniidazole. Concentrations indicated are amounts in 10 ml. of medium.
.2 0
2,O
082 0,02 VITAMIN 812 mpg./ml.
METHIONINE IO~g/ml.
Fig. 2.-Inhibition of growth of E. coli 113-3 with 4niercaptoben7imidazole compounds and reversal by vitamin Biz. Cultures of E . coli 113-3were grown as in Fig. 1. They contained concentrationsof 4-SH-benzimidazolesand vitamin BIZor methionine as indicated.
the growth of E. coli 113-3, but whose action was not a t all or only slightly reversed by increased vitamin Bl2or methionine.
Growth of the pyrimidine requiring mutant E . coli 15T- was inhibited by the compounds listed in Table IV. The action of some of these was reversed by increased concentrations of thymine. The pattern of inhibition and reversal was different from that with the vitamin B12requiring organism inhibited by the nitro or mercapto compounds. The most effective compounds were those substituted with methyl or chloro on 5- and/or 6-positions. The 5,6-dichlorobenzimidazoleshowed a pattern of inhibition and reversal which is graphed in Fig. 4. Inhibition of E . coli 15T- by 4-nitro-, B-chloro-
0
TABLE I1
CONCENTRATIONS OF SUBSTITUTED BENZIMIDAZOLES PRODUCING 50y0 INHIBITION OF GROWTHOF E. co2i 113-3 REQUIRING VITAMINB12 OR METHIONINE Code no. 2
48 66
63 148 91 73 54 33 38
SO2
SH SH NO2 hTO*
KO2
c1
NH2
..
C1 SHz NO2
1,3 8, 10, GO 5, 10 ... 10,20 NH2 10,20 S O z N f I ~17,35 KO2 40 NO2 50 NO2 50
7, 12,23 100 200 60 65 120 100 220 200
10,13 300 500 70 65 125 120 230 260
130
.. 90 100 270 320 200 200 210
TABLEI11 COSCENTRAITON OF SUBSTITUTED BENZIMIDAZOLES PRODUCISG 5070 INHIBITION O F GROWTHO F E . Coli 113-3 Concn. of drug. ra./ml.
Code no. 00 18 44 17 43 68 42 55 28 57 u 88 1 16
Position of substituent 5 6 2 4
..
C1
C~HP
..
..
CHI
..
.. .. .. .. .,
CIHS . ,
.,
..
xn2
20
Concn. of drug, rg./ml. Vitamin BIZ, mpg./ml. Methionine 10 pgiml. 0.02 0.2 2.0
Position 4 5 6
..
C1
CHa
.
c1
CHI CHI C1 CHI ,.
NIh .. .. CI ., NH2 .. Unsubsti tuted CHI . . CHI
.
c1 CHI CHz ,
.
..
CI NHz NIII SH
..
Vitamin R L ~mrg./ml. , 0.02 0.2 2.0 40 75 80 20 50 100, 150 300 250,150 100 500, 000 200
.. 9500
50 65
70 50
Methionine 10 pg./ml. 65
..
..
.. ..
140
150
..
170
..
150 300 300
150 300 200
..
..
150 300 250 200 400 500 450
400 300 500 >500
400 200 350 >500
..
..
5- 40 k
m -
I
60 bo
80
100 0.15
15 THYMINE ps/ml.
15
Fig. 4.-Reversal of 5,6-dichlorobenzimidazole inhibition of growth of E . coli l5T- by increased thymine. Concentrations of 5.6-dichlorobenzimidazoleand thymine as indicated are amounts per ml. of medium.
benzimidazole was not reversible by increased thymine but partially reversed b y methionine. Compounds which are substituted on the 2-position, that is the carbon of the imidazole ring, were inactive against the thymine requiring organism.
D. B. MCNAIRSCOTT, M. L. ROGERS, AND C. ROSE
CONCENTRATION O F
2
4
....
..............
..
....
..............
....
.............. ..............
1
17 44 7 25 68 190 63
....
L
a
TABLE IV BENZIMIDAZOLES PRODUCING 5070 INHIBITION O F E . Coli 15T- REQUIRINGTHYMINE
Position of substituent
Code no.
48 30 55 26 66 38 li2
SCBSTITUTED
....
..............
Ribose ....
.............. ..............
.... .... ....
.... .... ....
CHI c1 CH, OCHI
..
..
Concn. of drug, rg./ml. Thymine pg./ml. 0.15 1.5
G
CHs c1
.. .. ..
.. c1
c1 c1
SH
SO.?
..............
..
..
..
..
.............. ..............
KO2 NH2 NH2
.. ..
c1
..
c1
..
IiHt
.............. .............. ..............
....
5
..
SH
c1
...
..
NHz
.. NO2 .. .............. .. CHs .. Benzyl Methylpyridinium Estimated from preliminary screening. Partially reversed by methionine.
0.01-30 2.5 I, 12 1,15, 100 20 30 60 140 80,140 70,170 200b 200b 200b 300' 300'
Reversa1
80-120 60 > 100 5-500,600 190 30
7-
+ + + T
-
650 80,140 500 ...
i-, M a I
...
TABLE V CONCENTRATIONS OF SUBSTITUTED BENZIMIDAZOLES PRODUCING 50% OF E. coli B96 REQUIRING PURINE . . INHIBITION Position of substituent 4 5
Code no.
2
213
Dimer on butane
18
..
C4He
48
38 33
90 "', 1 3
CH,
NO*
.,
..
44
....
,
....
.
NH2
....
c1
....
.
..
KO2
148
I
SO?
..
c1 NO2
.. ..
..
E,
Concn. of drug (pg./ml.) Adenine-SO4 Guanine-HC1 5 pg./ml, 20 pg./ml. 5 pg./ml. 20 pg./ml.
..
1 10 20b
