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The influence of chemical structure on fungal activity. IV. The effect of bisphenolic-type compounds
The Influenceof ChemicalStructureon FungalActivity. IV. The Effect of Bisphenolic-TypeCompounds R. ReeceCorey’and Harold G. Shirk From the Department of Bacteriology, University of Maryland, CollegePark, Maryland, and the Prevention of Deterioration Center, National ResearchCouncil, Washington, D. C. Received November 1, 1954 INTRODUCTION
The antimicrobic properties of bisphenolic compoundsand their practical use to prevent mildew has already been discussedand referred to by Cade (I), Marsh, Greathouse, Butler, and Bollenbacher (2)) Marsh and Butler (3), Spaulding and Bondi (4), Brook (5), Moorehead (6), Abrams (7), and Marsh, Butler, and Clark (8). The large collection of bisphenols at the Prevention of Deterioration Center, National Research Council, made it possibleto test other variously substituted phenols as potential mildewproofing agents and to study the influence of chemical structure and substitution. The data for 24 bisphenols and related compoundsare presented and discussedin this report. METHODS The methods and testing procedures used in this study are essentially the same as those previously described in detail in the first report of this series by Shirk, Poelma, and Corey (9). In brief, a modified Caapek’s agar containing various concentrations of the chemical under investigation was inoculated with spores of AsperqilZus niger. After a 96-hr. incubation period, the inhibition of mycelial development was determined by comparing the growth with that taking place in a comparable time in the control. RESULTS AND DISCUSSION
Table I showsthe relative fungistatic potency of 13 bisphenolswhich have an unsubstituted methylene bridge. The results for the three iso1Present address: Department of Bacteriology, University of California, Davis, California. 196
FUNGAL
ACTIVITY.
TABLE of A. niger Growth Efected
Inhibition =
197
IV
I by Methvlene-Bridged ‘er cent inhibition
No.
CGbmvounds of grm vth
(10-A Ml , - - - -1 5.0 2.5 1.0 - -_
Concentration
Conm. to
inhibit yrowth 50%
10-r ‘U
1
84
76
70
54
37
2.4
2
62
58
48
39
27
5.0
3
58
50
40
28
15
7.5
100
97
89
74
66
65
,61 57
52
0.7
54
51
50
49
15
5.0
13
11
7
3
1
77
70
/64 57
52
d-cH2-Hb
4
Cl
Cl 5
Cl
OH
HO
Cl
CI>-C&-o Cl Cl
Cl OH
HO
Cl
O-CH?-c>
6
Cl HO
Cl
Cl
Cl
Cl
Cl
OH
Clo-CHzaCl
7
Cl
-
Br
>lO.O
Cl
(+&Hz-“QI>
8
0.4
Br
0.7
198
R.
REECE
COREY
AND
TABLE
HAROLD
G.
SHIRK
I-Continued P er cent inhibition -
No.
Compound
Concentration
of growth Concn.
-
-
1
-
to
inhibit growth 50%
(lo-’ Y) 1.0 -
IO-’ Y
Br
OH
HO
Br
C>-CH*-a
9
Br
O,N
OzN
51
50
50
48
42
9
8
6
5
2
>lO.O
36
31
25
16
10
>lO.O
26
25
20
14
7
>lO.O
>lO.O
5.0
NO,
1HOC>--CHzaOH -
16
Br
d-CH2-Hb
13
18
Br
Br
12
23
OH
10
11
26
Br
Br Br
29
NOz -
-
- - - - - - - - -
merit methylenediphenols (compounds l-3) show the effect of various positioning of the two hydroxyl groups on potency. Although a greater number of isomers were unavailable to permit a more comprehensive study, introduction of hydroxyl groups ortho to the bridge appears to be the most desirable configuration for inhibiting the growth of this fungus, The early findings of Bechold and Ehrlich (10) demonstrate that substitution of chlorine in the simple monophenols, particularly to the extent of introducing three halogen atoms, very significantly enhances microbicidal potency of the parent phenol. The data in Table I illustrate that limited chlorination increases the potency and inhibitory properties of 2,2’-methylenediphenol. The compound formed by introducing one chlorine atom in each phenolic grouping, now commonly
FUNGAL
ACTIVITY.
IV
199
known as G-42 or 2,2’-methylenebis[4-chlorophenol] (compound 4)) has significantly greater fungistatic properties than the parent diphenol. Additional chlorination results in compounds 5 and 6; compound 6 (G-l 1)2 is the bacteriostat now used in many soap and toilet preparations. The added chlorine in these chemicalsdetractsfrom the fungistatic potency as compared with that of G-4 as indicated by an increase in LDsa values (the interpolated molar concentrations at which growth is inhibited 50 %). Moreover, there is seemingly another mode of toxic action indicated by the different slope of the dosage-response curves for these two compounds. Monobromination of the basic diphenol (compound 1) resulting in the structure of compound 8 decreased fungistatic activity in contrast to monochlorination. The derivative formed by bromination with two atoms per phenolic grouping (compound 9) results in less effectiveness than the homologous chlorinated derivative. Despite the effectiveness of p-nitrophenol in protecting leather against mildew, it is interesting to note that both of the bisphenols formed by reacting either p-nitroor o-nitrophenol with formaldehyde had negligible fungistatic activity. The observed relationship between fungitoxicity and chemical structure corroborates the general results and conclusions of Marsh and Butler (3) who employed other test measures and procedures. Compounds such as 5, 6, 7, 9, 10, and 11 in Table I have structures which could cause a steric hindrance of the polar and active -OH group, and such configurations have low orders of fungistatic activity. Shirk (11) has already discussed and reported upon these phenomena with the monophenols in a separate report. Because of the pronounced insecticidal activity of dichlorodiphenyltrichloroethane (DDT), it was considered desirable to include in this study a series of trichloroethylidene-bridged bisphenols and to assay their fungistatic potency. Table II illustrates the activity of such compounds all possessing the same type of bridge and varying only in the substituents of the ring structures. Within the range of concentration used in our tests, compound 16 (DDT), despite its effectiveness against insects, has negligible effect on the growth and development of A. niger. The removal of either one or both chlorine atoms para to the bridge in this molecule leads to compounds (14 and 15 in Table II) which are more fungitoxic. Compound 14 has a relatively flat dosage-response curve which is similar to that of G-5 and G-11 (compounds 5 and 6, Table I) which contain more than two atoms of chlorine in each ring. 2 Registered
trade-mark
of Sindar
Corp.,
New York.
TABLE
Funaistatic
Activitu
II
. . Trichloroethvlidene-Bridaed lp
Compounds
of
‘er cent
=
inhibition
Concentration 00
7.5
of growtl -__ (10-r
5.0 -
2.5 -
.W
1
Concn. inhibit /yowth
to SOL&
1.0 10-4 M
0-Q
14
54
53
52
50
46
15
24
22
20
20
19
>lO.O
16
7
4
4
2
0
>lO.O
1
2
2
1
1
>lO.O
72
’70
62
46
30
3.1
10
10
5
2
2
Do 1100
00
lo
- - -
-
-
2.5
CClJ
BrD-LaBr cc13
*oa-L&-JoH cc13
200
>lO.O
0.2
FUNGAL
TABLE
ACTIVITY.
201
IV
II--Continued
-1Per cent inhibition of growth Concn.
NO.
Compound
l21
-
to
inhibit :rowth 500/o
tion (10~4M) 1.0 -
to-4 .lf
26
13.0
22
23
19
>lO.O
23
3
2
>lO.O
-
The only para substituent which increases the activity of this basic structure is the hydroxyl group (compound 18). The activities of compound 18 and compound 2 (Table I) are essentially the same, from which it might be inferred that the difference in bridge structure does not affect the toxicity of the molecule. Compound 20 is bhe trichloroethylidene-bridged bisphenol which is homologous with G-4 and which is markedly inhibitory. The LD60 is 20 X 10W6M or about 8 p.p.m. in contrast to 40 X lO-‘j M or 11 p.p.m. for G-4. As already noted with the methylene-bridged diphenols, further substitution of chlorine or other groups adjacent to the hydroxyl decreased the fungistatic potency; the nitro group (compound 23) decreased the activity more than either the chloro (compound 21) or bromo (compound 22) group. Unfortunately, additional comparable diphenols with and without the chlorinated bridge were not available to establish a firmer conclusion on the relative potency of methylene- and trichloroethylidene-bridged bisphenols.
202
R.
REECE
COREY
AND
HAROLD
G.
SHIRK
effect of three different type bridged bisphenols on A. niger FIG. 1. Inhibitory growth over a 500-fold range of concentration.
Figure 1 presents the results of a more detailed evaluation of the two most effective bisphenols (compounds 4 and 20) and includes as well a thiodiphenol which is identical except for the bridge structure. From the data plotted in Fig. 1, an evaluation is possible of the three bridge CCL I structures, -CHz-, -S---, and -CH-, \
FUNGAL ACTIVITY.
IV
203
ACKNOWLEDGMENTS We are indebted to S. I. Gertler, United States Department of Agriculture, and W. S. Gump, Givaudan-Delawanna, Inc., for supplying samples of the compounds used in this investigation. SUMMARY
A total of 24 compounds of a basic bisphenolic structure were tested for fungistatic potency by the nutrient agar dish technique using Aspergillus niger as the test organism. The most effective compounds contained two atoms of chlorine para to the hydroxyl groups with the bridge substituted in the 2,2’-position. Any deviation from this structure such as formation of a salt, esterification, or additional substitution in the ring structures diminished the potency of the compound. The trichloroethylidene bridge structure, linking two molecules of p-chlorophenol in the 2,2’-position, was the most effective compound studied. Further applicatory testing of this molecule is indicated. REFERENCES 1. CADE, A. R., Soap Sanit. Chemicals
20, 111 (1944). A., BUTLER, M. L., AND BOLLESBACHER, K., V. S. Dept. Agr., Tech. Bull. No. 892 (1945). MARSH, P. B., AND BUTLER, M. L., Ind. Eng. Chem. 38, 701 (1946). SPACJLDING,E. H., AND BONDI, A., JR., J. Infectious Diseases 80, 194 (1947). BROOK, E. I., Tret. Med. 44, 179 (1949). MOOREHEAD, C. A., North Am. F’eterinarian 29, 649 (1948). ABRAMS, E., Natl. Bur. Standards (U. S.), Misc. Publ. 188, 1 (1948). MAHSH, P. B., BUTLER, M. L., AND CLARK, B. S., Ind. Eng. Chem. 41, 2176 (1949). SHIHK, H. G., POELMA, P. L., AND COREY, R. R., JR., Arch. Biochem. and Biophys. 3!2, 386 (1951). BECIIOLD, H., AND EHRLTCH,P., 2. physiol. Chem. 47, 173, (1906). SHIR.K, H. G., Arch,. Biochem. and Biophys. 61, 258 (1954). GOLDSWORTHY,M.C., AND GERTLER, S. I., Plant Disease Rptr. Suppl. 182, 89 (1949).
2. MARSH,P.B.,GREATHOUSE,G. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
The antimicrobic properties of bisphenolic compoundsand their practical use to prevent mildew has already been discussedand referred to by Cade (I), Marsh, Greathouse, Butler, and Bollenbacher (2)) Marsh and Butler (3), Spaulding and Bondi (4), Brook (5), Moorehead (6), Abrams (7), and Marsh, Butler, and Clark (8). The large collection of bisphenols at the Prevention of Deterioration Center, National Research Council, made it possibleto test other variously substituted phenols as potential mildewproofing agents and to study the influence of chemical structure and substitution. The data for 24 bisphenols and related compoundsare presented and discussedin this report. METHODS The methods and testing procedures used in this study are essentially the same as those previously described in detail in the first report of this series by Shirk, Poelma, and Corey (9). In brief, a modified Caapek’s agar containing various concentrations of the chemical under investigation was inoculated with spores of AsperqilZus niger. After a 96-hr. incubation period, the inhibition of mycelial development was determined by comparing the growth with that taking place in a comparable time in the control. RESULTS AND DISCUSSION
Table I showsthe relative fungistatic potency of 13 bisphenolswhich have an unsubstituted methylene bridge. The results for the three iso1Present address: Department of Bacteriology, University of California, Davis, California. 196
FUNGAL
ACTIVITY.
TABLE of A. niger Growth Efected
Inhibition =
197
IV
I by Methvlene-Bridged ‘er cent inhibition
No.
CGbmvounds of grm vth
(10-A Ml , - - - -1 5.0 2.5 1.0 - -_
Concentration
Conm. to
inhibit yrowth 50%
10-r ‘U
1
84
76
70
54
37
2.4
2
62
58
48
39
27
5.0
3
58
50
40
28
15
7.5
100
97
89
74
66
65
,61 57
52
0.7
54
51
50
49
15
5.0
13
11
7
3
1
77
70
/64 57
52
d-cH2-Hb
4
Cl
Cl 5
Cl
OH
HO
Cl
CI>-C&-o Cl Cl
Cl OH
HO
Cl
O-CH?-c>
6
Cl HO
Cl
Cl
Cl
Cl
Cl
OH
Clo-CHzaCl
7
Cl
-
Br
>lO.O
Cl
(+&Hz-“QI>
8
0.4
Br
0.7
198
R.
REECE
COREY
AND
TABLE
HAROLD
G.
SHIRK
I-Continued P er cent inhibition -
No.
Compound
Concentration
of growth Concn.
-
-
1
-
to
inhibit growth 50%
(lo-’ Y) 1.0 -
IO-’ Y
Br
OH
HO
Br
C>-CH*-a
9
Br
O,N
OzN
51
50
50
48
42
9
8
6
5
2
>lO.O
36
31
25
16
10
>lO.O
26
25
20
14
7
>lO.O
>lO.O
5.0
NO,
1HOC>--CHzaOH -
16
Br
d-CH2-Hb
13
18
Br
Br
12
23
OH
10
11
26
Br
Br Br
29
NOz -
-
- - - - - - - - -
merit methylenediphenols (compounds l-3) show the effect of various positioning of the two hydroxyl groups on potency. Although a greater number of isomers were unavailable to permit a more comprehensive study, introduction of hydroxyl groups ortho to the bridge appears to be the most desirable configuration for inhibiting the growth of this fungus, The early findings of Bechold and Ehrlich (10) demonstrate that substitution of chlorine in the simple monophenols, particularly to the extent of introducing three halogen atoms, very significantly enhances microbicidal potency of the parent phenol. The data in Table I illustrate that limited chlorination increases the potency and inhibitory properties of 2,2’-methylenediphenol. The compound formed by introducing one chlorine atom in each phenolic grouping, now commonly
FUNGAL
ACTIVITY.
IV
199
known as G-42 or 2,2’-methylenebis[4-chlorophenol] (compound 4)) has significantly greater fungistatic properties than the parent diphenol. Additional chlorination results in compounds 5 and 6; compound 6 (G-l 1)2 is the bacteriostat now used in many soap and toilet preparations. The added chlorine in these chemicalsdetractsfrom the fungistatic potency as compared with that of G-4 as indicated by an increase in LDsa values (the interpolated molar concentrations at which growth is inhibited 50 %). Moreover, there is seemingly another mode of toxic action indicated by the different slope of the dosage-response curves for these two compounds. Monobromination of the basic diphenol (compound 1) resulting in the structure of compound 8 decreased fungistatic activity in contrast to monochlorination. The derivative formed by bromination with two atoms per phenolic grouping (compound 9) results in less effectiveness than the homologous chlorinated derivative. Despite the effectiveness of p-nitrophenol in protecting leather against mildew, it is interesting to note that both of the bisphenols formed by reacting either p-nitroor o-nitrophenol with formaldehyde had negligible fungistatic activity. The observed relationship between fungitoxicity and chemical structure corroborates the general results and conclusions of Marsh and Butler (3) who employed other test measures and procedures. Compounds such as 5, 6, 7, 9, 10, and 11 in Table I have structures which could cause a steric hindrance of the polar and active -OH group, and such configurations have low orders of fungistatic activity. Shirk (11) has already discussed and reported upon these phenomena with the monophenols in a separate report. Because of the pronounced insecticidal activity of dichlorodiphenyltrichloroethane (DDT), it was considered desirable to include in this study a series of trichloroethylidene-bridged bisphenols and to assay their fungistatic potency. Table II illustrates the activity of such compounds all possessing the same type of bridge and varying only in the substituents of the ring structures. Within the range of concentration used in our tests, compound 16 (DDT), despite its effectiveness against insects, has negligible effect on the growth and development of A. niger. The removal of either one or both chlorine atoms para to the bridge in this molecule leads to compounds (14 and 15 in Table II) which are more fungitoxic. Compound 14 has a relatively flat dosage-response curve which is similar to that of G-5 and G-11 (compounds 5 and 6, Table I) which contain more than two atoms of chlorine in each ring. 2 Registered
trade-mark
of Sindar
Corp.,
New York.
TABLE
Funaistatic
Activitu
II
. . Trichloroethvlidene-Bridaed lp
Compounds
of
‘er cent
=
inhibition
Concentration 00
7.5
of growtl -__ (10-r
5.0 -
2.5 -
.W
1
Concn. inhibit /yowth
to SOL&
1.0 10-4 M
0-Q
14
54
53
52
50
46
15
24
22
20
20
19
>lO.O
16
7
4
4
2
0
>lO.O
1
2
2
1
1
>lO.O
72
’70
62
46
30
3.1
10
10
5
2
2
Do 1100
00
lo
- - -
-
-
2.5
CClJ
BrD-LaBr cc13
*oa-L&-JoH cc13
200
>lO.O
0.2
FUNGAL
TABLE
ACTIVITY.
201
IV
II--Continued
-1Per cent inhibition of growth Concn.
NO.
Compound
l21
-
to
inhibit :rowth 500/o
tion (10~4M) 1.0 -
to-4 .lf
26
13.0
22
23
19
>lO.O
23
3
2
>lO.O
-
The only para substituent which increases the activity of this basic structure is the hydroxyl group (compound 18). The activities of compound 18 and compound 2 (Table I) are essentially the same, from which it might be inferred that the difference in bridge structure does not affect the toxicity of the molecule. Compound 20 is bhe trichloroethylidene-bridged bisphenol which is homologous with G-4 and which is markedly inhibitory. The LD60 is 20 X 10W6M or about 8 p.p.m. in contrast to 40 X lO-‘j M or 11 p.p.m. for G-4. As already noted with the methylene-bridged diphenols, further substitution of chlorine or other groups adjacent to the hydroxyl decreased the fungistatic potency; the nitro group (compound 23) decreased the activity more than either the chloro (compound 21) or bromo (compound 22) group. Unfortunately, additional comparable diphenols with and without the chlorinated bridge were not available to establish a firmer conclusion on the relative potency of methylene- and trichloroethylidene-bridged bisphenols.
202
R.
REECE
COREY
AND
HAROLD
G.
SHIRK
effect of three different type bridged bisphenols on A. niger FIG. 1. Inhibitory growth over a 500-fold range of concentration.
Figure 1 presents the results of a more detailed evaluation of the two most effective bisphenols (compounds 4 and 20) and includes as well a thiodiphenol which is identical except for the bridge structure. From the data plotted in Fig. 1, an evaluation is possible of the three bridge CCL I structures, -CHz-, -S---, and -CH-, \
FUNGAL ACTIVITY.
IV
203
ACKNOWLEDGMENTS We are indebted to S. I. Gertler, United States Department of Agriculture, and W. S. Gump, Givaudan-Delawanna, Inc., for supplying samples of the compounds used in this investigation. SUMMARY
A total of 24 compounds of a basic bisphenolic structure were tested for fungistatic potency by the nutrient agar dish technique using Aspergillus niger as the test organism. The most effective compounds contained two atoms of chlorine para to the hydroxyl groups with the bridge substituted in the 2,2’-position. Any deviation from this structure such as formation of a salt, esterification, or additional substitution in the ring structures diminished the potency of the compound. The trichloroethylidene bridge structure, linking two molecules of p-chlorophenol in the 2,2’-position, was the most effective compound studied. Further applicatory testing of this molecule is indicated. REFERENCES 1. CADE, A. R., Soap Sanit. Chemicals
20, 111 (1944). A., BUTLER, M. L., AND BOLLESBACHER, K., V. S. Dept. Agr., Tech. Bull. No. 892 (1945). MARSH, P. B., AND BUTLER, M. L., Ind. Eng. Chem. 38, 701 (1946). SPACJLDING,E. H., AND BONDI, A., JR., J. Infectious Diseases 80, 194 (1947). BROOK, E. I., Tret. Med. 44, 179 (1949). MOOREHEAD, C. A., North Am. F’eterinarian 29, 649 (1948). ABRAMS, E., Natl. Bur. Standards (U. S.), Misc. Publ. 188, 1 (1948). MAHSH, P. B., BUTLER, M. L., AND CLARK, B. S., Ind. Eng. Chem. 41, 2176 (1949). SHIHK, H. G., POELMA, P. L., AND COREY, R. R., JR., Arch. Biochem. and Biophys. 3!2, 386 (1951). BECIIOLD, H., AND EHRLTCH,P., 2. physiol. Chem. 47, 173, (1906). SHIR.K, H. G., Arch,. Biochem. and Biophys. 61, 258 (1954). GOLDSWORTHY,M.C., AND GERTLER, S. I., Plant Disease Rptr. Suppl. 182, 89 (1949).
2. MARSH,P.B.,GREATHOUSE,G. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.