Mildew-Preventing Activity of Rhodanine Derivatives KETONE DERIVATIVES FRANCES C. BROWN, CHARLES K. BRADSHER, AND ELISE N. LAWTON Department of Chemistry, Duke University, Durham, N . C .
I
T IS generally known that cotton fabrics when used or stored under warm, humid conditions may suffer damage or destruction because of mildew and rot. Such damage is usually manifested by changes in appearance of the textile and by loss in tensile strength. This deterioration appears to result largely from the action of cellulolytic fungi and bacteria (19)on the cotton fibers and is particularly prevalent where the fibers come in contact with soil. While certain chemical agents (16) are known to arrest the growth of mildew, it seemed desirable to find new and perhaps more effective compounds for this purpose. Among the common organic sulfur compounds used as fungicides ( I S , 14, 17) may be included the ferric and zinc salts of dimethyldithiocarbamic acid (I), tetramethylthiuramdisulfide (11), tetramethylthiurammonosulfide (111),and disodium ethylenebisdithiocarbamate (IV).
S
/I
(( CHa h N - C S
(CH8)zN-
)2Zn
I
S
II
(CH~)zN-C-S-C-N(
B
i
4-SI1
CH3)2
I11 S
H
NaSC-N-CHzCH2NII I IV
H
S
' e
SNa
All of these compounds contain the structure -N-C-Sand an examination of other organic substances containing the same structure seemed desirable. I
I
i
Rhodanine (V) which contains the group -N-C-Sas a part of a ring structure has been patented as a fungicide ( I ) , but the tests reported in this paper indicate that it is not effective in protecting cotton cloth against the attack of Chaetomium globosum, a cellulolytic fungus, or against the organisms present in soil. The purpose of the present investigation was to determine whether suitable changes in the structure of rhodanine would produce compounds of greater fungicidal and mildew-proofing activity. It has been known for some time (18) that rhodanine undergoes condensation with aldehydes to yield products of Type VI (R' = H).
I !
Such compounds retain the toxiphoric group -N-C-Sand, in addition, have a carbonyl group conjugated with a double bond, a feature present in the fungicidal chalcones and acrylophenones (12). Recently it has been shown that the conden-
sation is general for methyl and alicyclic ketones ( 6 ) , and the mildew-preventing activity of these new condensation products (VI) forms the subject of the present communication. 0
0
R
V
HN-C
//
VI EXPERIMENTAL
Twenty-five compounds of Type VI were screened for mildewproofing activity. The screening process involved pure culture and soil burial tests performed on replicate cotton twill strips impregnated with 2y0 of the candidate fungicide. The tests were incubation with pure cultures of Chaetomium globosum, soil burial for 2 weeks, and soil burial for 4 weeks. The compounds were prepared by the condensation of rhodanine or 3-substituted rhodanines with the appropriate ketone (6,6 ) . The testing procedure was a modification of the ASTM standard method (8). The test fabric was dyed cotton herringbone twill (8.5-ounce O.D. No. 7), which was impregnated with a 2% concentration of the candidate fungicide by the wet-weight pickup method. A steel die was used to cut the cloth into strips 6 inches (warp) by 1 inch (fill). PURE CULTURE TESTS. The unsterilized strips were placed on beds of sterile mineral salts agar in French square bottles with ventilated caps, inoculated with 1 ml. of spore suspension, and incubated a t 30" C. for 14 days. Five bottles were inoculated with spores of Chaetomium globosum. The spores were from standard test microorganisms supplied by the Medical Division of the Chemical Warfare Service. At the end of the 14-day period, the extent of growth was observed and the strips were washed with warm water, allowed to dry a t room temperature, and tested for tensile strength with a Scott tester. Ten strips of untreated cloth were broken for comparison and the per cent loss in strength of the treated strips was recorded. In each pure culture test, control samples of untreated cloth were inoculated with spores of Chaetomium globosum. After 2 weeks' incubation a t 30" C., the controls were completely covered with fungal growth and had lost all tensile strength. The culture medium in the above tests consisted of: ammonium nitrate, 3 grams; potassium dihydrogen phosphate, 2.5 grams; magnesium sulfate heptahydrate, 2 grams; agar, 15 grams; distilled water, 1 liter. The pH of the medium was adjusted to 5.2 i 0.2 b y addition of 0.1 N hydrochloric acid.
1(127
INDUSTRIAL AND ENGINEERING CHEMISTRY
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I. PERFORMAXCE IE; MILDEW-RESISTANCE TESTSO F SOME ALIPHATICKETONEDERIVATlVES O F RIiODANINE HN,,C=O CHs
TABLE
I
Compound
/
Rating, Chaetomlium slobosum
R
SO.
/
223 233 247 262 364 245 261 239 285
0 0 0 0
324
Strength Loss, % Soil Burial Culture 2 4 (2 weeke) weeks weeks 0 2 0 0 0 0 0 4 92
65 84 0 10 3 6 4 64 100
100 100 48 58 36 42 20 96 100
0
0
86
100
0
0
47
54
74
96
34
100
1 0 0 0.2 4.8
332 325
-
C
H
HN-CO
t!
255
I / c=c
'y
S
z a CzHi
'CzHs
I n the visual observation of the extent of growth of the microorganism, the approximate percentage of cloth covered is indicated in the tables by the following 0-5 rating system:
data for the condensation products of methyl aliphatic ketones with rhodanine. Table I1 presents similar information for the alicyclic ketone derivatives. Table I11 shows the effect of replacing the hydrogen atom in the 3-position of some compounds listed in Table I1 by methyl, allyl, or phenyl groups. Table IV contains data for comparable mildew-proofing tests on various substances which have been used or patented as fungicides. Different species of fungi differ markedly in their resistance to the presence of certain chemicals (15). Not all of these substances have been recommended as mildew-proofing agents and failure of any compound in these tests does not reflect upon its suitability for other purposes where fungicidal action is required. The results are included merely to show the relative effectiveness of rhodanine derivatives as cellulose preservatives by comparison with other types of organic molecules. RELATION OF FUNGICIDAL ACTIVITY TO CHEMICAL STRUCTURE
From a comparison of the data in Tables I through 111, it will be seen that a t the concentration employed the majority of the compounds protected cloth in the pure culture tests with Chaetomium globosum. It appears evident that soil burial is a more drastic test of the protection which a candidate fungicide affords cloth than the pure culture tests. Block ( 3 ) has presented data which indicate that under the conditions of his experiments the results of labo-
TABLE11. PERFORMANCE I N MILDEW-RESISTANCE TESTSOF BLICYCLIC KETONEDERIVATIVES OF RHODANINE "-CEO S=C
I
I
C=R \
Rating 0 1
2 3 4 5
Coverage, 70 No growth 0-10 10-20 20-50 50-70 70-100
Vol. 45, No. 5
\/
/
S
Compound
284
H2-cr R
NO.
=c?
Rating, Chaetomzum globoszsm
0
Strength Loss, % Soil Burial Culture (2 weeks) 2 weeks 4 wecks
0
9
33
\CHz-cH2 SOILBURIALTESTS. Ten strips containing 2% by weight of the candidate CHz-CHI / fungicide were buried to a depth of 4.5 225 =C 'CH2 0 0 27 33 inches in flats containing moist, com'CHz-CHI / posted greenhouse soil maintained a t /CHn-cH2 approximately 30" C. Five replicate 248 =C \CH* 6 0 0 0 strips were removed a t the end of 2 / 'CH-CHI weeks and five a t the end of 4 weeks. The strips %-ere washed and dried as dH3 in the pure culture tests and their tenCHz-CHz \ sile strength was measured. The per ", 16 0 283 CH2 84 cent loss in strength was determined by 'CH2-CH / comparison with untreated cloth. \cH3 Since samples of soil differ greatly CHz-CHz in their destructive action on cloth and / since any given soil sample may differ 244 =C \CHCHs 0 1 4 9 / in activity as moisture content varies, 'CHs-CHI parallel tests were run on cloth strips CH3 impregnated with 2% chloranil. I n \ 331 =C CH--b-CzHs 1.4 ' 20 45 97 61 determinations, the average percentage loss in strength of 2% chloranil\CHz-cHa/ bHs impregnated samples after soil burial for 14 days was 83.6%. If the chlorani1 reference sample showed less than 70% loss in strength, the soil burial tests were reratory soil burial tests are, in general, in agreement with outdoor peated. exposure tests, and that deterioration from soil burial for 2 weeks The results of the experiments (the average of five determinais the same as that from outdoor exposure in shaded areas for 2 tions) are assembled in Tables I through IV. Table I gives the years. Consequently, i t would appear that the protection
=c(
May 1953
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INDUSTRIAL AND ENGINEERING CHEMISTRY
toxic action is greater than the amount of substance which can reach the site RHODANINE DERIVATIVES of activity. R’N-C=O From the data, in Table 11, it is evident that certain alicyclic ketone derivb=R atives of rhodanine are good protectants for cotton cloth. However, the S Strength Loss, % number of carbon atoms attached to Rating, ComChaetomium Culture pound the cyclohexane ring (compounds 331 R’ globosum ( 2 weeks) 2 weeks 4 weeks R No. and 244) and their location with respect to attachment to the rhodanine -CHa 0 0 91 100 357 ring (compounds 248, 283, and 244) are important factors in determining the degree or absence of protection. While 1 77 100 -CHzCH=CHz 0.2 the introduction of a methyl group in 386 the para position of the cyclohexyl ring increases activity, a tertiary amyl group in the same position lowers the 60 100 100 5 372 activity as compared with the unsubstituted cyclohexylidene derivative. A methyl group in the meta position of --CHI 0.2 0 93 100 355 the cyclohexylidene ring lowers activity, while the same group in the ortho or para position increases the protective power and produces com-CHzCH=CHz 0.2 2 80 99 385 pounds which are extremely effective against the cellulolytic organisms in soil. 6 65 100 100 375 I n several studies of the relation of chemical structure to biological activity. a resemblance between the activity of alicyclic compounds and aliphatic compounds which, by coiling afforded during soil burial for 4 weeks is a good index of the rotof the carbon chains, can have the same skeletal configuration has been observed (8). Several of the ketone derivaresisting activity of the compounds in question. The data in Table I afford interesting information concerning tives reported in this study afford the basis for such a the effect of tpe length of side chain on preservative activity. comparison. If in compounds of Type VI (R’ = CHI) the residual strength of samples after exposure to soil for 4 weeks is plotted against the number of carbon atoms in R, where R is limited to normal alkyl groups, the curve in Figure 1 is obtained. The lower members of the series are not effective, but beginning with the propyl group there is a marked increase in activity. This protective action reaches its maximum with the amyl group and 5-(a-n-amylethylidene)rhodanine, erroneously reported as a-n-amylhexylidenerhodanine in (4), is one of the most active mildew-preventing compounds considered in this report. I t s performance in these tests compares favorably with that of many of the compounds listed in Table IV. When more than five carbon atoms are present in the normal alkyl group, activity drops markedly and with nine carbon atoms there is almost no protection in soil burial or even in pure culture tests. It has been noted with several homologous series that the lower members show slight biological activity and that as the carbon chain becomes longer, the activity increases to a maximum and then drops off more or less rapidly. Such peaks of activity against gram-negative bacteria are shown by the alkylated phenols and resorcinols (20) and b y the aliphatic bases (11). The methyl ketone derivatives of rhodanine follow the same pattern. Various theories have been proposed to account for the peak of activity which is found with increasing number of carbon I atoms. Most of the theories assume that as the number of carbon atoms increases there is increasing toxicity and decreasing availability of the compound. The decreasing availability has been attributed to decreasing water solubility (9),to increas1 2 3 4 5 6 7 8 9 ing micelle formation which limits the number of single toxic NUMBER OF CARBON ATOMS IN R molecules (7), or to inability of the larger molecule to penetrate the cell membrane (IO). I n each case the drop in activity would Figure 1. Effect of Length of Side Chain on be caused by the fact that the concentration required for Preservative Activity
TABLE 111.
PERFORMANCE I N MILDEW-RESISTANCE TESTS O F SOME
3,6-DISUBSTITUTED
a
“
=+
“
I
--- -- --
INDUSTRIAL AND ENGINEERING CHEMISTRY
1030
TABLEITr.
PERFORRlANCE I N
Compound
Compound Name 9,lO-Phenanthrapuinone 2,2’-Dihydroxy-o,5’-dichlorodiphenylmethane Salicyl anilide Rhodanine 8-Hydroxyquinoline Sodium diethyldithiocarhamate Tetramethylthiuramdisulfide 2,4-Thiaaolidinedione 2-Mercaptobenzothiaz?le Cetyltrimethylammoniuni bromide Pentachlorophenol 2- (Pentachloroplienoxy) ethanol
NO.
120 log 186 191 211 212 216 224 232 242
256 268
MILDEW-RESISTANCE TESTSO F Rating, Chaetomaun
Culture
( 2 weeks)
globosum
Strength Loss, % Soil Burial 2 weeks ’ 4 weeks 0 1
0
0
5
0
0 100 0
66 0 100 89
100 69 100 100
0
0
43
100
0
0 29 2
0 100 88
100
9 1
69
0
13
100 0 43
2.4
0 0 0
Compounds 247, 255, and 284 have the same number of carbon atoms and similar structures. Likewise, compounds 364
R=
JH3 /CH3
CHz-CH3 R=C’
\CH2-CH3
‘CH1--CHt 284
255
CHs
/ R=C \
CHr-CHz
R=C /
‘CH*--CH? 247
CH3
R=C
AHz
/
FUNGICIDES
0
0
-
VARIOUS
\
FH*
CHg-CH2 CHr-CH2 364 226 ( R represents the rhodanine nucleus)
and 225 have similar arrangements of carbon atoms. Comparison of the data in Tables I and I1 shows that with the exception of compound 255, which is not so effective as its related compounds, there is a striking similarity in the biological activity between the “open” and the “closed” forms. The deleterious effect of increased length of carbon chain, regardless of whether part of the molecule is present in an alicyclic or aliphatic structure, is shown by compounds 285 (Table I ) and 331 (Table 11). From the results in Tables I1 and I11 it may be seen that the excellent protection afforded by 5-cyclohexylidenerhodanine or 5-(p-methylcyclohexylidene)rhodanine may be reduced or lost if the hydrogen in the 3-position of the rhodanine nucleus is replaced by alkyl or aryl groups. All of the 3,5-disubstituted rhodanines included in this report would be useless for protec-
0
58 100
Vol. 45, No. 5
tion in soil burial, and of this group the 3-phenyl derivatives are ineffective even in the pure culture tests. It seems evident that the size and shape of the molecule as well as the presence of toxic groups are important factors in determining the protective power of mildew-proofing agents. Further correlations between the variations in structure of this interesting group of compounds and biological activity will form the basis of subsequent reports. However, many factors other than biological activity determine whether a given agent can be applied commercially as a cellulose preservative. LITERATURE CITED
(1) Alvord, E. B. (to Grasselli Chemical Co.), U. S.Patent 1,962,109 (June 5, 1934). (2) American Society for Testing iwaterials, “ASTM Standards,” Part 111-A, pp. 1098-1104, Philadelphia, Pa., 1946. (3) Block, S.S.,IA-D. ESG. CHEM.,41, 1783 (1949). (4) Brown, F. C., and Bradsher, C. K., Nature, 168, 171 (1931). (5) Brown, F. C., Bradsher, C. K., Bond, S.hl., and Potter, M., J . Am. Chem. SOC.,73, 2357 (1951). (6) Brown, F. C., Bradsher, C. X., McCallum, S. G., and Potter, M.. J . Oru. Chem.. 15. 174 (1960). (7) Danielli, J. I?., “Cell Physiology and Pharmacology,” New York, Elsevier Publishing Co., 1950. (8) Dodds, E. C., Goldberg, L., Lawson, W.,and Robinson, R., Nature, 141, 247 (1938). (9) Ferguson, J., Proc. Rag. Soc. (London),127B, 387 (1939). (10) Finholt. R. W.. Weeks. PI..and Hathawas. C.. IXD.ENG. CHEM.,44, 101 (1952). (11) Fuller, A. T., Biochem. J . , 3 6 , 548 (1942). 67, 112 (1945). (12) Geiger, W.B., and Conn, J., J . A m . Chem. SOC., (13) Horsfall, J. G., “Fungicides and Their Action,” Waltham, Mass., Chronica Botanica Co., 1946. (14) Klopping, H. L., and Kerk, G. J. M. van der, Rec. trav. chim., 70, 917, 949 (1951). (15) Lilly, V. G., and Barnett, H. L., “Physiology of the Fungi,” pp. 245-65. New York. McGraw-Hill Book Co.. 1951. (16) Marsh, P. B., Tertile Research J., 17, 597 (1947). (17) bliller, C. R., and Elson, W. O., J . Bact., 57, 47 (1949). (18) Nencki, RI.,Ber., 17, 2277 (1884). (19) Siu, R. G. H., “Microbial Decomposition of Cellulose with Special Reference to Cotton Textiles,” New York, Reinhold Publishing Corp., 1951. (20) Suter, C. &I., Chem. Revs., 28, 269 (1941). R E C E I ~ Efor D review June 14,1952. ACCEPTED September 13, 1952 This work was supported by a contract with the Medical Division, Chemical Corps, U. S. Army.
(Mildew-Preventing Activity of Rhodanine Derivatives)
SOME 5-ARYLIDENE DERIVATIVES FRANCES C. BROWN, CHARLES I
