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Copper Complexes of Aromatic Dithiocarbamates and Their Antifungal Activity*
Copper Complexes of Aromatic Dithiocarbamates and Their Antifungal Activity* By WILLIAM 0. FOYE, IVAN B. VAN de WORKEEN, Jr.,t and JOSEPH D. MATTHES Phenyl and 2-pyridyl dithiocarbamate salts and esters have been prepared and found
to form stable corn lexes, probably chelates, with copper (11) ion. The aromatic dithiocarbamates sgowed a low order of antifungal and antibacterial activity, but
their copper complexes were practically inactive. This finding does not support previous postulations that antifungal dithiocarbamates exert their fungitoxicity as toxic copper chelates.
NVESTICATIONS at the
duPont Company in 1931 showed t h a t derivatives of dithiocarbamic acid possessed antifungal and antibacterial properties, and a patent reporting the antifungal activity of thiuram disulfides was issued to Tisdale and Williams ( I ) in 1934. The activity of several dithiocarbamic acid derivatives against fungi pathogenic t o man was noted b y Hall in 1938 in a n unpublished report, and was later studied b y Miller and Elson ( 2 ) . Within twenty years of their discovery, the dithiocarbamates had become widely used for controlling plant pathogens (3). The suggestion that metal chelation is a mechanism of biotoxic action for antifungal agents was made by Zentmyer (4) in 1943. He observed t h a t the fungicidal action of oxine (8hydroxyquinoline) was reversed b y zinc ion, and at low p H , where the chelating ability of oxine was weak, the oxine showed weak fungicidal activity. Shortly after, Albert ( 5 ) demonstrated conclusively t h a t the bactericidal action of oxine is due to the formation in the cell of a toxic 1: 1 chelate with iron or copper. The copper chelate of oxine has subsequently faund commercial use mildew-proofing textiles a n d paint. Goksgyr (6) has also postulated t h a t the fungicidal action of sodium dimethyldithiocarbamate is due t o the formation of toxic 1 : 1 chelates with copper and zinc. T o test further the hypothesis t h a t formation of toxic metal chelates is a mechanism of fungicidal action, a series of dithiocarbamate salts and esters has been prepared in order t o compare the activities of chelated and nonchelated derivatives. Aromatic dithiocarbamates were selected for this purpose and also t o determine the relative
I
*
Received February 13, 1958, from the Massachusetts College of Pharmacy, Boston. Abstracted from the thesis submitted by I. B. Van d e Workeen, Jr.. as a requirement for the degree of blaster of Science, June 195fi. i Present address: Chemistry Department. Rosion University. Mass. The authors are indebted to Drs. K. K. Chen and D. Pleming of the Lilly Research Laboratories for conducting the antimicrobial tests and furnishing the results.
fungicidal properties of aromatic dithiocarbamates, which have not been looked at to any extent for fungicidal effects.
METHODS OF PREPARATION The ammonium salt of phenyldithitxarbaniic acid was easily prepared by the method of Dains, Brew, esters were obtained by ster, and Olander ( i ) and standard procedures. Use of this method to obtain 2-pyridyldithiocarbamates was unsuccessful, however, and a review of the literature showed that heterocyclic dithiocarbmates have not received much attention. In a review of the chemistry of dithiocarbamates by Chabrier and Nachmias (8), for instance, no heterocyclic derivatives were mentioned. Only two references found in the literature described the preparation of pyridyl derivatives, and in only one of these was the product isolated, but not characterized. This product was a mixture believed to contain the 4-aminopyridyl salt of 4pyridylditliiocarbamic acid (9). in analogy t o the general formation of aliphatic dithiocarbamates. Among the methods attempted for the preparation of 2-pyridyldithiocarbamic acid derivatives was that of Camps (9) for the suspected salt of the 4isomer, using 2-aminopyridine, carbon disulfide, and ethanol, and also that of Klarer and Behnisch (10). who mentioned the intermediate formation of the potassium salt of 2-pyridyldithiocarbatnic acid. Both of these methods failed, as did two procedures in which aniline has been used to obtain phenyldithiocarbamic acid. The latter two required the use of alcoholic potassium hydroxide or aqueous ammonia in the presence o f carbon disulfide and the aromatic aniine. The desired reactbin was accxnplished by first forming the sodium salt o f 2-arninopyridine in nonaqueous media with sodaniide, according to Chichibabin (111, and the isolated sodium salt was then suspended in benzene and allowed to react with carbon disulfide. The product was found by analysis t o be the 2-aminopyridyl salt o f 2-pyridytdithiocarbamic acid, which is analogous t o the product described by Camps (9) from 4-aminopyridine. Esters of this acid, which were obtained by reaction of the salt with organic halides, were difficult to purify but did give nitrogen analyses approximating theoretical. The dithiocarbarnate salts and esters prcpnred and their properties are listed in Table I The reaction o f copper ion with the salts and esters described above was carried out to determine the
556
SCIENTIFIC EDITION
August 1958
557
TABLEI.-AROMATIC DITHIOCARBAMATES
R
Ar
2-Pyridyl 2-Pyridyl 2-Pyridyl Phenyl Phenyl Phenyl
Yield, % .-
M. P."
2-Pyridylammonium Ethyl p-Nitrophenyl Ammonium Ethyl P-Nitrophenyl
...
86
Oil 128-1S0' 92-93 ' 58-60 ' 88-90 O
CiiHizN4Sz CdWWz CizHgNaOzS2 C7HioNzSz CPHiiNSz CiaHioNzOzSZ
50
10 73 41 50
Yob-
----Analysis, Calcd.
Formula
Found
C:49.98 H: 4.60 N:14.14 N: 14.18 N:15.06 N: 7.11 N: 9.62
50.41 5.27 13.87 13.67 14.80 7.21 9.48
a The melting points were taken on a Fisher-Johns block and are uncorrected. b The carbon-hydrogen analysis was obtained from the Weiler and Strauss Microanalytical Laboratory, Oxford, England. The nitrogen analyses were done by the Kjeldahl procedure, using selenized Hengar granules as catalyst.
possibility of complex or chelate formation. This was done in a suitable solvent with aqueous copper sulfate, and in each case complexing was indicated by a marked drop in pH, and formation of a colored precipitate in the case of the salts. Copper complexes soluble in the organic media used were obtained from the esters. Analysis indicated that complexes having a 2 :1 ratio of dithiocarbamic acid to copper were obtained from the salts and the pnitrophenyl esters, and complexes with a 1: 1 ratio were obtained from the ethyl esters. The physical properties of the complexes prepared are listed in Table 11. The structures of 1: 1 metal chelates of dithiocarbamic acids have been determined by Chatt, et al. (12), and pictured as four-membered rings (structure I) with several resonating forms possible. The 2 : l copper complexes can accordingly be represented by structure 11, although open-chain complexes with the copper coordinated to two sulfurs or possibly two nitrogens are also possible and would also cause a drop in pH on formation. Complexing by means of the pyridine nitrogen is not postulated for these compounds, since the phenyl derivatives gave the same results as the pyridyl.
\.1c u
I
I1
degree of activity differed slightly in several cases, these compounds generally showed activity toward the same organisms, and both were also negative toward a number of other organisms. The copper chelates of these acids, on the other hand, showed essentially no activity toward the test organisms. Only U. avenue was inhibited by the chelates a t a concentration of 200 mcg./ml. but not a t 50 mcg./ml. (The esters and their copper derivatives have not as yet been tested.) This result implies that dithiocarbamates do not exert their fungicidal action in the form of toxic copper chelates, although chelation of essential copper in the organisms is still a plausible mechanism of action. Further such comparisons should be made with other dithiocarbamates, however, although the comparative results might be obscured somewhat with weaker chelators or stronger antifungal agents.
BIOLOGICAL. RESULTS Antifungal and antibacterial tests were carried out a t the Lilly Research Laboratories, and the results of positive activity are shown in Table 111. It is apparent that the salts of phenyl- and 2pyridyldithiocarbamic acids have essentially the same activity against microorganisms; while the
TABLEII.-COPPER COMPLEXES OF THE DITHIOCARBAMATES
sI
(Ar-N=C--SR),Cu Ar
2-Pyridyl 2-Pyridyl 2-Pyridyl Phenyl Phenyl Phenyl a
R
n
pH Dropa pH Yield, Units % '
2 H CZH~ 1 CEH~NO~2 H 2 CZH~ 1 C6H4N02 2
6.6 3.4 2.0 3.0 3.0 2.5
74 40 48 84 56 50
M. P.b
Color
115'(dec.) 88-90' 132-134O llOO(dec.) 50-52' 103-105"
Green-black Green-black Yellow-green Yellow Yellow-green Red
Formula
ClzHloN4SlCu 13.93 13.42 C~H~ONZSZCU 9.40 9.94 CuHl&04s4Cu 12.96 11.76 CirHizNeSXu 7.00 6.96 CpHlIN~Cu.2Hz0 4.71 5.10 CZSHIBN~OIS~CU 8.67 8.53
The drop in pII was recorded during the addition of copper sulfate.
6 The melting points were taken on a Fisher-Johns block and are uncorrected.
N Analysis, % c Calcd. Found
The nitrogen analyses were done by the Kjeldahl procedure, using selenized Hengar granules as catalyst.
558
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
TDLE III.-ANTWICROBIAL TESTINGOF DITHIOCARBAMATE SALTS‘ Test Organism
Staphylococcus albus Bacillus subtilis Mycobacterium tuberculosis Klebsiella pneumoniae Trichophyton interdigitale Shigella paradysenteriae Xanthomonas phaseoli Monilinia fructicola Ustilago avenae Alternaria solani Fusarium monilijorme Colletotrichum gossypii Ascochyta imperfecta Sclerotinia bataticola
THE
Inhibitory Concn., mcg./ml.b Phenyl 2-Pyridyl Deriv.c Deriv.d
200 50
50 100 100 ... 25 100 12.5 <6.25 200 50 25 50
200 50 50 100 100 25 50 12.5 <6.25 100 200 50
Carried out at the Lilly Research Laboratories by D. Fleming. b The agar dilution technique was used, the bacteria being observed for forty-eight hours. and the bacterial and fungal plant pathogens being observed for seventy-two hours. Those organisms having no inhibitory levels indicated were not a6ected by 200 mcg./ml. C Ammonium phenyldithiocarbamate. d 2-Pyridylammonium 2-pyridyldithiocarbamate.
EXPERIMENTAL Ammonium Pheny1dithiocarbamate.-The procedure of Dains, Brewster, and Olander (7) was used. A %yoyield of white powder was obtained which melted at 92-93’. Losanitsch (13) first described this compound as decomposing at 100”. Dains, et al., list no physical constants. Ethyl Pheny1dithiocarbamate.-Three grams (0.016 mole) of ammonium phenyldithiocarbamate and 75 ml. (1 mole) of ethyl bromide were stirred a t room temperature for four hours. The mixture was filtered, and the filtrate was distilled under reduced pressure. The remaining semisolid was taken up in ether and allowed to crystallize, giving 1.25 Gm. (41%) of yellow-green crystals; m. p. 5860’. j-Nitrophenyl Pheny1dithiocarbamate.-One gram (0.005 mole) of ammonium phenyldithiocarbamate was dissolved with gentle heating in 30 ml. of distilled water. Ethyl alcohol (20 ml.) and p nitrochlorobenzene (1.0 Gm., 0.006 mole) were added, and the solution was refluxed for one hour. A light yellow powder precipitated after the solution cooled, giving 0.8 Gm. (50%) of product melting at 88-90 ’. 2-Pyridylammonium 2-Pyridy1dithiocarbaate.A solution of 12 Gm. (0.31 mole) of sodamide in 50 ml. of benzene was treated dropwise with 25 Gm. (0.27 mole) of 2-aminopyridine dissolved in 175 ml. of benzene with warming. The mixture was refluxed for two hours and allowed to cool. A solution of 17 ml. (0.28 mole) of carbon disulfide in 50 ml. of benzene was then added dropwise with stirring, and the resulting solution was refluxed with stirring (mercury seal) for four hours and allowed t o stand overnight. The yellow precipitate which appeared
Vol. XLVII, No. 8
was washed with ether and dried. The yield was 30 Gm. (86%). Erratic results were obtained from Kjeldahl determinations on this product using either selenium or mercuric oxide as catalyst, but a carbon-hydrogen combustion analysis gave values approaching theoretical. The product was contaminated with sodium ion, however. Ethyl 2-Pyridyldithiocarbaate.-Three grams (0.011 mole) of the 2-pyridyldithiocarbamate salt and 75 ml. (1 mole) of ethyl bromide were stirred at room temperature for four hours. The mixture was filtered, and the filtrate was distilled under reduced pressure. The red, oily liquid remaining was dissolved in ether, which was partially evaporated. A yield of 1.5 Gm. (68%) of red oil was obtained which decomposed on attempted distillation under reduced pressure. p-Nitrophenyl 2-Pyridyldithiocarbamate.-One gram (0.005 mole) of the 2-pyridyldithiocarbamate salt was dissolved with gentle heating in 30 ml. of distilled water. Ethyl alcohol (20 m l . ) and p nitrochlorobenzene (10 Gm., 0.006 mole) were added, and the solution was refluxed for one hour. A green powder precipitated after the solution cooled, giving 0.15 Gm. (10%) of product melting at 128-130”. Chelation with Copper (II) Ion.-The following procedure is representative. To a filtered solution of 10 Gm. (0.05 mole) of ammonium phenyldithiocarbamate in 75 ml. of water was added gradually, with stirring, a solution of 4 Gm. (0.025 mole) of copper sulfate in 50 ml. of water. During the reaction the pH dropped from 8.3 to 5.3. A yellow precipitate was collected, washed with water, and allowed to dry a t room temperature. The yield was 9.0 Gm. (84%); and the product decomposed at 110’. I n the corresponding reaction with the esters, an organic solvent was used-acetone for the ethyl esters and ethanol for the p-nitrophenyl esters. After treatment with aqueous copper sulfate solution in which a minimum amount of water was used, the unreacted copper sulfate was removed by filtration. The filtrate was then allowed t o evaporate t o dryness to yield the complexes.
REFERENCES (1) Tisdale, W. H., and Williams, I., CT. S. pat. 1,972,961 (1934). (2) Miller, 0. R., and Elson, W. O., J . Racteriol., 57, 47(1949). (3) Horsfall, J. G., “Principles of Fungicidal Action,” Chronica Botanica Co., Waltham, Mas$., 1956, p. 171. (4) Zentmyer, G. A . , Pkylagalhalagy, 33, 112(1913); Science, 100, 294(1944). (5) Albert, A . , Gibson, M. I., and Rubbo, S. D., &if. J . Expll. Palhol., 34, 119(1953). (6) GoksZyr, J., Naftrre, 175, 820(1955). (7) Dains, F. B.,,, Brewster, R. Q., and Olander, C. P., “Organic Syntheses, Coll. Vol. I, 2nd ed., John Wiley and Sons, Inc., New York, 1941, p. 447. (8) Chabrier, P.,and Nachmias, G., Bttll. IOC. chim. Froncr, 17D 51(1950). (9) Cambs, R., Arch. Pharm., 240,345(1902). (10) Klarer, J., and Behsisch, R., German pat. 832,891
\____,.
(14.53)
(11) Chichibabin, A. E.. Konovalova, R. A,, and Konovalova, A. A,, Ber., 54,814(1921). (12) Chatt. I . , Duncanson, I,. A,, and Venanzi. L. M . , Noltrre 177 1042(105(3). (13)’I,osktsch, S. M., B w . , 24, 3021(1891).
to form stable corn lexes, probably chelates, with copper (11) ion. The aromatic dithiocarbamates sgowed a low order of antifungal and antibacterial activity, but
their copper complexes were practically inactive. This finding does not support previous postulations that antifungal dithiocarbamates exert their fungitoxicity as toxic copper chelates.
NVESTICATIONS at the
duPont Company in 1931 showed t h a t derivatives of dithiocarbamic acid possessed antifungal and antibacterial properties, and a patent reporting the antifungal activity of thiuram disulfides was issued to Tisdale and Williams ( I ) in 1934. The activity of several dithiocarbamic acid derivatives against fungi pathogenic t o man was noted b y Hall in 1938 in a n unpublished report, and was later studied b y Miller and Elson ( 2 ) . Within twenty years of their discovery, the dithiocarbamates had become widely used for controlling plant pathogens (3). The suggestion that metal chelation is a mechanism of biotoxic action for antifungal agents was made by Zentmyer (4) in 1943. He observed t h a t the fungicidal action of oxine (8hydroxyquinoline) was reversed b y zinc ion, and at low p H , where the chelating ability of oxine was weak, the oxine showed weak fungicidal activity. Shortly after, Albert ( 5 ) demonstrated conclusively t h a t the bactericidal action of oxine is due to the formation in the cell of a toxic 1: 1 chelate with iron or copper. The copper chelate of oxine has subsequently faund commercial use mildew-proofing textiles a n d paint. Goksgyr (6) has also postulated t h a t the fungicidal action of sodium dimethyldithiocarbamate is due t o the formation of toxic 1 : 1 chelates with copper and zinc. T o test further the hypothesis t h a t formation of toxic metal chelates is a mechanism of fungicidal action, a series of dithiocarbamate salts and esters has been prepared in order t o compare the activities of chelated and nonchelated derivatives. Aromatic dithiocarbamates were selected for this purpose and also t o determine the relative
I
*
Received February 13, 1958, from the Massachusetts College of Pharmacy, Boston. Abstracted from the thesis submitted by I. B. Van d e Workeen, Jr.. as a requirement for the degree of blaster of Science, June 195fi. i Present address: Chemistry Department. Rosion University. Mass. The authors are indebted to Drs. K. K. Chen and D. Pleming of the Lilly Research Laboratories for conducting the antimicrobial tests and furnishing the results.
fungicidal properties of aromatic dithiocarbamates, which have not been looked at to any extent for fungicidal effects.
METHODS OF PREPARATION The ammonium salt of phenyldithitxarbaniic acid was easily prepared by the method of Dains, Brew, esters were obtained by ster, and Olander ( i ) and standard procedures. Use of this method to obtain 2-pyridyldithiocarbamates was unsuccessful, however, and a review of the literature showed that heterocyclic dithiocarbmates have not received much attention. In a review of the chemistry of dithiocarbamates by Chabrier and Nachmias (8), for instance, no heterocyclic derivatives were mentioned. Only two references found in the literature described the preparation of pyridyl derivatives, and in only one of these was the product isolated, but not characterized. This product was a mixture believed to contain the 4-aminopyridyl salt of 4pyridylditliiocarbamic acid (9). in analogy t o the general formation of aliphatic dithiocarbamates. Among the methods attempted for the preparation of 2-pyridyldithiocarbamic acid derivatives was that of Camps (9) for the suspected salt of the 4isomer, using 2-aminopyridine, carbon disulfide, and ethanol, and also that of Klarer and Behnisch (10). who mentioned the intermediate formation of the potassium salt of 2-pyridyldithiocarbatnic acid. Both of these methods failed, as did two procedures in which aniline has been used to obtain phenyldithiocarbamic acid. The latter two required the use of alcoholic potassium hydroxide or aqueous ammonia in the presence o f carbon disulfide and the aromatic aniine. The desired reactbin was accxnplished by first forming the sodium salt o f 2-arninopyridine in nonaqueous media with sodaniide, according to Chichibabin (111, and the isolated sodium salt was then suspended in benzene and allowed to react with carbon disulfide. The product was found by analysis t o be the 2-aminopyridyl salt o f 2-pyridytdithiocarbamic acid, which is analogous t o the product described by Camps (9) from 4-aminopyridine. Esters of this acid, which were obtained by reaction of the salt with organic halides, were difficult to purify but did give nitrogen analyses approximating theoretical. The dithiocarbarnate salts and esters prcpnred and their properties are listed in Table I The reaction o f copper ion with the salts and esters described above was carried out to determine the
556
SCIENTIFIC EDITION
August 1958
557
TABLEI.-AROMATIC DITHIOCARBAMATES
R
Ar
2-Pyridyl 2-Pyridyl 2-Pyridyl Phenyl Phenyl Phenyl
Yield, % .-
M. P."
2-Pyridylammonium Ethyl p-Nitrophenyl Ammonium Ethyl P-Nitrophenyl
...
86
Oil 128-1S0' 92-93 ' 58-60 ' 88-90 O
CiiHizN4Sz CdWWz CizHgNaOzS2 C7HioNzSz CPHiiNSz CiaHioNzOzSZ
50
10 73 41 50
Yob-
----Analysis, Calcd.
Formula
Found
C:49.98 H: 4.60 N:14.14 N: 14.18 N:15.06 N: 7.11 N: 9.62
50.41 5.27 13.87 13.67 14.80 7.21 9.48
a The melting points were taken on a Fisher-Johns block and are uncorrected. b The carbon-hydrogen analysis was obtained from the Weiler and Strauss Microanalytical Laboratory, Oxford, England. The nitrogen analyses were done by the Kjeldahl procedure, using selenized Hengar granules as catalyst.
possibility of complex or chelate formation. This was done in a suitable solvent with aqueous copper sulfate, and in each case complexing was indicated by a marked drop in pH, and formation of a colored precipitate in the case of the salts. Copper complexes soluble in the organic media used were obtained from the esters. Analysis indicated that complexes having a 2 :1 ratio of dithiocarbamic acid to copper were obtained from the salts and the pnitrophenyl esters, and complexes with a 1: 1 ratio were obtained from the ethyl esters. The physical properties of the complexes prepared are listed in Table 11. The structures of 1: 1 metal chelates of dithiocarbamic acids have been determined by Chatt, et al. (12), and pictured as four-membered rings (structure I) with several resonating forms possible. The 2 : l copper complexes can accordingly be represented by structure 11, although open-chain complexes with the copper coordinated to two sulfurs or possibly two nitrogens are also possible and would also cause a drop in pH on formation. Complexing by means of the pyridine nitrogen is not postulated for these compounds, since the phenyl derivatives gave the same results as the pyridyl.
\.1c u
I
I1
degree of activity differed slightly in several cases, these compounds generally showed activity toward the same organisms, and both were also negative toward a number of other organisms. The copper chelates of these acids, on the other hand, showed essentially no activity toward the test organisms. Only U. avenue was inhibited by the chelates a t a concentration of 200 mcg./ml. but not a t 50 mcg./ml. (The esters and their copper derivatives have not as yet been tested.) This result implies that dithiocarbamates do not exert their fungicidal action in the form of toxic copper chelates, although chelation of essential copper in the organisms is still a plausible mechanism of action. Further such comparisons should be made with other dithiocarbamates, however, although the comparative results might be obscured somewhat with weaker chelators or stronger antifungal agents.
BIOLOGICAL. RESULTS Antifungal and antibacterial tests were carried out a t the Lilly Research Laboratories, and the results of positive activity are shown in Table 111. It is apparent that the salts of phenyl- and 2pyridyldithiocarbamic acids have essentially the same activity against microorganisms; while the
TABLEII.-COPPER COMPLEXES OF THE DITHIOCARBAMATES
sI
(Ar-N=C--SR),Cu Ar
2-Pyridyl 2-Pyridyl 2-Pyridyl Phenyl Phenyl Phenyl a
R
n
pH Dropa pH Yield, Units % '
2 H CZH~ 1 CEH~NO~2 H 2 CZH~ 1 C6H4N02 2
6.6 3.4 2.0 3.0 3.0 2.5
74 40 48 84 56 50
M. P.b
Color
115'(dec.) 88-90' 132-134O llOO(dec.) 50-52' 103-105"
Green-black Green-black Yellow-green Yellow Yellow-green Red
Formula
ClzHloN4SlCu 13.93 13.42 C~H~ONZSZCU 9.40 9.94 CuHl&04s4Cu 12.96 11.76 CirHizNeSXu 7.00 6.96 CpHlIN~Cu.2Hz0 4.71 5.10 CZSHIBN~OIS~CU 8.67 8.53
The drop in pII was recorded during the addition of copper sulfate.
6 The melting points were taken on a Fisher-Johns block and are uncorrected.
N Analysis, % c Calcd. Found
The nitrogen analyses were done by the Kjeldahl procedure, using selenized Hengar granules as catalyst.
558
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
TDLE III.-ANTWICROBIAL TESTINGOF DITHIOCARBAMATE SALTS‘ Test Organism
Staphylococcus albus Bacillus subtilis Mycobacterium tuberculosis Klebsiella pneumoniae Trichophyton interdigitale Shigella paradysenteriae Xanthomonas phaseoli Monilinia fructicola Ustilago avenae Alternaria solani Fusarium monilijorme Colletotrichum gossypii Ascochyta imperfecta Sclerotinia bataticola
THE
Inhibitory Concn., mcg./ml.b Phenyl 2-Pyridyl Deriv.c Deriv.d
200 50
50 100 100 ... 25 100 12.5 <6.25 200 50 25 50
200 50 50 100 100 25 50 12.5 <6.25 100 200 50
Carried out at the Lilly Research Laboratories by D. Fleming. b The agar dilution technique was used, the bacteria being observed for forty-eight hours. and the bacterial and fungal plant pathogens being observed for seventy-two hours. Those organisms having no inhibitory levels indicated were not a6ected by 200 mcg./ml. C Ammonium phenyldithiocarbamate. d 2-Pyridylammonium 2-pyridyldithiocarbamate.
EXPERIMENTAL Ammonium Pheny1dithiocarbamate.-The procedure of Dains, Brewster, and Olander (7) was used. A %yoyield of white powder was obtained which melted at 92-93’. Losanitsch (13) first described this compound as decomposing at 100”. Dains, et al., list no physical constants. Ethyl Pheny1dithiocarbamate.-Three grams (0.016 mole) of ammonium phenyldithiocarbamate and 75 ml. (1 mole) of ethyl bromide were stirred a t room temperature for four hours. The mixture was filtered, and the filtrate was distilled under reduced pressure. The remaining semisolid was taken up in ether and allowed to crystallize, giving 1.25 Gm. (41%) of yellow-green crystals; m. p. 5860’. j-Nitrophenyl Pheny1dithiocarbamate.-One gram (0.005 mole) of ammonium phenyldithiocarbamate was dissolved with gentle heating in 30 ml. of distilled water. Ethyl alcohol (20 ml.) and p nitrochlorobenzene (1.0 Gm., 0.006 mole) were added, and the solution was refluxed for one hour. A light yellow powder precipitated after the solution cooled, giving 0.8 Gm. (50%) of product melting at 88-90 ’. 2-Pyridylammonium 2-Pyridy1dithiocarbaate.A solution of 12 Gm. (0.31 mole) of sodamide in 50 ml. of benzene was treated dropwise with 25 Gm. (0.27 mole) of 2-aminopyridine dissolved in 175 ml. of benzene with warming. The mixture was refluxed for two hours and allowed to cool. A solution of 17 ml. (0.28 mole) of carbon disulfide in 50 ml. of benzene was then added dropwise with stirring, and the resulting solution was refluxed with stirring (mercury seal) for four hours and allowed t o stand overnight. The yellow precipitate which appeared
Vol. XLVII, No. 8
was washed with ether and dried. The yield was 30 Gm. (86%). Erratic results were obtained from Kjeldahl determinations on this product using either selenium or mercuric oxide as catalyst, but a carbon-hydrogen combustion analysis gave values approaching theoretical. The product was contaminated with sodium ion, however. Ethyl 2-Pyridyldithiocarbaate.-Three grams (0.011 mole) of the 2-pyridyldithiocarbamate salt and 75 ml. (1 mole) of ethyl bromide were stirred at room temperature for four hours. The mixture was filtered, and the filtrate was distilled under reduced pressure. The red, oily liquid remaining was dissolved in ether, which was partially evaporated. A yield of 1.5 Gm. (68%) of red oil was obtained which decomposed on attempted distillation under reduced pressure. p-Nitrophenyl 2-Pyridyldithiocarbamate.-One gram (0.005 mole) of the 2-pyridyldithiocarbamate salt was dissolved with gentle heating in 30 ml. of distilled water. Ethyl alcohol (20 m l . ) and p nitrochlorobenzene (10 Gm., 0.006 mole) were added, and the solution was refluxed for one hour. A green powder precipitated after the solution cooled, giving 0.15 Gm. (10%) of product melting at 128-130”. Chelation with Copper (II) Ion.-The following procedure is representative. To a filtered solution of 10 Gm. (0.05 mole) of ammonium phenyldithiocarbamate in 75 ml. of water was added gradually, with stirring, a solution of 4 Gm. (0.025 mole) of copper sulfate in 50 ml. of water. During the reaction the pH dropped from 8.3 to 5.3. A yellow precipitate was collected, washed with water, and allowed to dry a t room temperature. The yield was 9.0 Gm. (84%); and the product decomposed at 110’. I n the corresponding reaction with the esters, an organic solvent was used-acetone for the ethyl esters and ethanol for the p-nitrophenyl esters. After treatment with aqueous copper sulfate solution in which a minimum amount of water was used, the unreacted copper sulfate was removed by filtration. The filtrate was then allowed t o evaporate t o dryness to yield the complexes.
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(11) Chichibabin, A. E.. Konovalova, R. A,, and Konovalova, A. A,, Ber., 54,814(1921). (12) Chatt. I . , Duncanson, I,. A,, and Venanzi. L. M . , Noltrre 177 1042(105(3). (13)’I,osktsch, S. M., B w . , 24, 3021(1891).