Ann. appl. Biol. (1964), 54, 115-127

Printed in Great Britoin ' I5

Investigations on fungicides XI. The antifungal activity of some derivatives of dithiocarbamic acid

BY G. A. CARTER, J. L. GARRAWAY, D. M. SPENCER AND R. L. WAIN Agricultural Research Council Plant Growth Substance and Systemic Fungicides Unit

and Department of Physical Sciences, Wye College, Ashford, Kent

(Received 12 March 1964)

S U M M A R Y

A series of P-(thiocarbamoy1thio)-aldehydes and -ketones, N-methyl analogues and z,3-dihydro-z-thio-1 ,s-thiazines derived therefrom were examined in laboratory tests for fungitoxicity against seven fungi and in greenhouse tests against four plant diseases. The most active compounds were the phenyl- or trichloromethyl-substituted P-(N-methylthiocarbamoy1thio)-ketones. The corresponding thiazines were less fungitoxic. Structure/fungitoxicity relationships are discussed with special reference to the influence of water solubility and water/oil partition coefficients.

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

Thiazines derived from dithiocarbamic acids are of interest as fungicides (Bluestone, 1960; Carter, Garraway, Spencer & Wain, 1963). 2,3-Dihydro-z-thio-1,3-thiazines have been reported in connexion with rubber vulcanization accelerator studies (Jansen & Mathes, 1955) and Owens (1959) has discussed the relationship between the biochemical effects and chemical properties of certain sulphur-containing rubber vulcanizing accelerators. In the present work the fungitoxic properties of q3-dihydro- z-thio-I ,3-thiazines and the intermediate products used in their formation were investigated.

The importance of a correct balance between lipoid and water solubilities of a fungicide has been established, with compounds possessing hydrocarbon chains attached to various toxiphores (Woodcock, 1961) or with widely different hetero- cyclic nuclei (Horsfall & Rich, 1951). The relationship between the number and positions of alkyl substituents attached to the thiazine ring and such physico-chemical properties as water solubility and partition coefficient has now been examined, Other studies have also been carried out to assess the effects of various substituent groups on ultra-violet absorption spectra and on the stability of these compounds in neutral, acidic and alkaline media (Garraway, 1964a, b).

8-2

I 16 G. A. CARTER AND OTHERS

MATERIALS

Structural formulae of the compounds included in this investigation are given below

(1) (11) (111)

Full details of the preparation of these compounds (1-1 I I) are available (Garraway, 1964a, b). The nature of the groups R,-R5 together with melting points and certain analytical data are given in Tables I and 2.

E X P E R I M E N T A L

Biological The anti-fungal activity of all compounds was assessed in spore germination tests;

separate tests for fumigant activity were also carried out. Compounds showing marked fungicidal activity in vitro were tested as protectants against diseases of three plant species.

Spore germination tests Since most of the compounds were of low water solubility they were tested by the

method described by Spencer (1962) with certain modifications.

(i) Elimination of compounds with low activity Sufficient of a stock solution of each compound in absolute alcohol was pipetted

on to cavity slides to give deposits of 10 and ~ o o p g . after evaporation of the solvent at 28" C. Conidia of Botrytis cinerea were washed from 10- to 14-day-old cultures, centrifuged, washed three times in distilled water and resuspended in 0.2 % sucrose solution at a density of approximately 20,000 spores per ml. 0.1 ml. of this spore suspension was then added to each cavity, thus providing concentrations of the test chemical, assuming complete solution, of IOO and 1000 ,ug./ml. After 18 hr. incubation in moist chambers at 25" C. the percentage spore germination was determined. Compounds which did not significantly reduce germination at 1000 ,ug./ml. were discarded at this stage. See Tables I and 2, Test B.

(ii) Further examination of active compounds This was carried out using the method of Carter et al. (1963). Duplicate samples

(0.1 ml.) of a series of concentrations of each compound in absolute alcohol were pipetted into polystyrene microbeakers and the solvent was allowed to evaporate at 28" C. Spore suspension (0.1 ml.), prepared as described above, was added and the slides were transferred to moist chambers. The percentage germination was assessed

Cod

e no. I

.

2 3 4 5 6 7 8 9 I

0

I1

I2

13

I4

I5

16

I7

I8

I9

20

21

22

23

24

25

26

27

Tab

le I

. Th

e an

tifun

gal a

ctiv

ities

of /

3-(thiocarbamoylthio)-aldehydes a

nd -k

eton

es (formula I

or

11 o

n p.

I I 6

) T

est B

f

Subs

titue

nts (

form

ula

I)

Rl

H

H

H

H

Me

Me

Me

Me

H

H

H

H

Me

H

Me

Me

Ph

H

Me

Me

Ph

Me

Me

Ph

Me

Me

Ph

R2

H

Me

H

Me

H

H

Me

H

H

Me

H

Me

H

H

H

Me

H

H

H

Me

H

H

Me

H

H

Me

H

R8

H

H

Me

Me

H

Me

Me

Me

H

H

Me

Me

Me

Ph

Ph

Ph

Ph

Ph

Ph

Ph

Ph

CC

l, C

Cl,

CC

l, cc

1,

CC

l, C

Cl,

R4

H

H

H

H

H

H

H

Me

H

H

H

H

Me

H

H

H

H

H

H

H H

H

H

H H

H

H

Rs H

H

H

H

H

H

H

H

Me

Me

Me

Me

Me

H

H

H

H

Me

Me

Me

Me

H

H

H

Me

Me

Me

F8.j

128-

129

97-9

8 I 16

-1 1

75

116-

117

65-9

011

126-

127

147-

1 48

13

3-13

49

101-1

02

95-9

6 110-1

11

76-7

7

137-

1389

16

811

135-

1361

1 11

8-11

9ll

7879

11

97-9

8 11

7-11

8 13

2-13

51]

152-

1531

1 121-1

22

88-8

9 Io

3-Io

qll

126-

127

1541

1

139-

1421

1

Ana

lysi

s*

Tes

t At

I

c

Fkm

d R

equi

red h

9'

42

8.56

8.

77

7.81

8.

61

8.21

7'

55

7'25

8.

51

8.0

2

7'71

7.

04

6.72

6.

01

5'99

5'

52

5'90

5'

25

5'24

4

66

5'

14

49

4

3'99

4'

50

40

5

48

9

45

8

9'39

8.

58

8.58

7.

89

8-58

7'

89

7'32

7'

32

8.58

7

49

7.

89

7'32

6-

83

6.22

5.

86

5'53

5.86

5'

53

5'24

4

44

4

99

4

75

4'

09

47

5

45

4

3'93

4'65

E

D

E

E A

E

B E

E E

E

E

C

E

D

D

E

E B B A

A

A

A

A

C

A

G

E

D

E

E C

E

D

E

E E

D

E

B

E

E

E

E

C

A

B

A

B

A

A

A

D

A

V

E D

E

D

A

D

E

E

E E

B E

A

E

E

D

E

E

A

A

C A

C

A

A

C

A

M

E E E

E

D

E

E

E

E E

B

E

A

E

E

E

B E

A

A

E B E

A

A

D

A

Ab E

E

E

E

E

E

E

E

E

E

E

E B

C

E

E

E

E

C

E

E

B

A

A

A

C

A

c E

E

E

D

C

D

C

E

E

E

D

E A

E

E

E

E

D

A

A

A

B

A

A

A

B

A

Slop

e of p

robi

i re

gres

sion

line

4-25

f 0.

50

-

-

-

-

-

2.81

f 0

21

5.

26fo

.58

8.70

f 1

.23

6-67

f 0.

50

2'1

2 f 0'

21

6.89

f 0

89

6.

67 f 0

89

-

-

ED

gd

and

s.%

fiduc

ial

limits

oL

g./m

l.)

> I

OO

O

> IO

OO

>

IOO

O

> I

OOO

> I

OO

O

> IO

OO

>

IO

OO

>

IOO

O

> 1

000

> I

OOO

> I

OO

O

> I

OO

O

540 f 5

0

> I

OO

O

> I

OOO

> IO

OO

>

1000

> I

OO

O

8i-

I 50f5

> I

OO

O

I52

f7

> IO

OO

3*

0+

01

8

1+

6

7If

5

63

f5

Nitrogen y

o.

t K

ey to

abb

revi

atio

ns: A

n, A

sper

gillu

s nig

er; G

, Glo

wel

la ci

ngul

ata;

V, V

ertic

illiu

m al

bo-a

trum

; M, M

onili

a fr

uctig

ena;

Ab,

Alte

rnur

ia b

rass

i-

f U

sing

Bot

rytis

cin

erea

. 5

Jans

en &

Mat

hes

(195

5) g

ive

the following

mel

ting

poin

ts:

3, 1

15-1

16'

C.;

8, 1

28-1

29'

c.; 1

5, 1

23-1

24'

C.;

17, 1

50-1

51'

C.

I1 M

eltin

g with d

ecom

posi

tion.

cico

la; C

, Cla

dosp

oriu

m cu

cum

erin

um.

A, c. 1

0 pg

./ml.;

B,

10-1

00

pg./

ml.

; C

, 100-500

pg

./m

l.; D

, 500-1000 p

g./m

l.; E

, >

1000 p

g./m

l.

s U

U

Cod

e no

.

28

29

30

31

32

33

34

35

37

39

40

41

42

43

44

45

47

49

50

51

52

53

36

38

46

48

Tab

le 2.

The antifungal activities of

2,3-dihydro-2-thio-1,g-thiazines (jo

rmul

a II

I on

p. I

16)

Tes

t BS -

ED

50

and

5%

Su

bstit

uent

s (fo

rmul

a 11

1)

Ana

lysi

s*

Tes

t At

fidu

cial

R,

Re

R,

R4

R,

("C

.) Fo

und

Req

uire

d A

n G

V

M

Ab

C

regr

essi

on li

ne

(pg

./m

l.)

H

H

H

H

H

106-

107

10

22

10

07

C

C

C

C

D

C

6.67

+_0.

63

633+

_75

H

Me

H

H

H

145-

146

9.62

9.

65

C

C

C

C

C

B

20

~0

0+

1~

36

676

+_1

6 H

H

M

eH

H

94

-95

9.76

9.

65

D

C

B

D

D

B

434+

0*&

20

8+16

H

M

e M

e H

H

11

5-11

6 8.

57

8.79

D

C

C

C

D

B

9.

80+1

.90

4325

17

Me

H

H

H

H

88-8

9 9'

59

9.65

D

D

C

C

D

B

6.

67k0

.63

679f

62

Me

Me

Me

H

H

130-

131

8.02

8.

08

D

D

D

E

E

D

-

Me

H

Me

Me

H

94-9

6s

8.29

8.

08

C

D

C

C

D

D

-

L

, >

m.p

. - f

, Sl

ope o

f pro

bit

limits

9

Me

H

Me

H

H

103-

104

8.53

8.

79

C

D

C

C

C

C

6.67

k0.8

9 51

4+35

** n

>

1000

> I

OO

O

H

H

H

H

Me

41-4

2.5

9.81

9.

65

D

D

C

D

D

D

4'17

f0.5

0 52

8548

H

M

e H

H

M

e 60

-61.

5 8.

71

8.80

D

D

C

C

C

C

15

.38k

1.93

70

7k17

H

H

M

e H

M

e 41

-42.

5 8.

63

8.80

D

D

C

D

D

D

5.

26k0

.89

664+

61

H

Me

Me

H

Me

104-

105

8.18

8.

09

E

D

C

C

E

C

-

> 1000

Me

H

Me

Me

Me

58-5

9 7.

63

7.48

C

C

C

D

D

C

5.

13+

041

527k

36

> IO

OO

H

H

Ph

H

H

13

4-13

5 6.

83

6.76

E

E

E

E

E

E

-

> Io

oo

Me

H

Ph

H

H

147-

1485

6.

27

6.33

E

E

E

E

E

D

-

Me

Me

Ph

H

H

183-

184l

l 6.

14

5.96

E

E

E

E

E

E

-

> 1

000

> IO

OO

Ph

H

Ph

H

H

12

6-12

76

5.07

4.

94

E

E E

E

E

D

-

> X

OO

O

H

H

Ph

H

Me

101--102

6.35

6.

33

E

E E

E E

E

-

> I

000

Me

H

Ph

H

Me

80.5

-81.

5 6.

14

5.96

C

E

C

C

E

E

-

> I

000

Me

Me

Ph

H

Me

162-

163

5.47

5.

62

E

E

E

E E

C

-

> I

000

Me

H

CC

l, H

H

17

3-17

4 5.

19

5.33

B

C

C

C

C

B

-

Me

Me

CC

l, H

H

95

-96

5'24

5-

06

B

C

B

B C

C

8

70

k1

.48

11

5+5

Ph

H

CC1,

H

H

120-121

4.15

4

31

B

A

A

C

E

A

-

Me

H

cc1,

H

Me

101-1

02

5'22

5.

06

B

C

C

C

C

B

8.70

+07

5 14

2+7

Me

Me

CC

l, H

M

e 64

-66

4.89

4-

82

D

D

D

E

E

C

-

Ph

H

CC

I9

H

Me

166-

1681

1 4.

32

4.13

A

A

A

B

B B

-

3 8 El:

> I

000

> I

QOO

> I

OO

O

U

U

00

9, t,

1, a

nd II

as T

able

I.

5 Ja

nsen

& M

athe

s (19

55) give

the

follo

win

g m

eltin

g po

ints

: 35,

96-

97'

C.;

42,

145"

C.;

44, 1

25-1

26"

C.

Investigations on fungicides. XI 119

after 18 hr. at 25" C. All the active compounds within a chemical series were tested at the same time, first over a wide range of concentrations selected on the results obtained in the preliminary test described above. On the following day a narrower concentration range of freshly prepared solutions was examined. The ED 50 values and regression slopes, together with their errors, were obtained by the method of Litchfield & Fertig (1941) and are given in Tables I and 2, Test B.

(iii) Activity against spores of several fungi The method similar to that described in (i) was employed using spores of Aspergillus

niger, Verticillium albo-atrum, Monilia fructigena, Alternaria brassicicola and Clado- sporium cucumerinum and a concentration range extended to include 10 and 500 pg./ml. After incubation at 25" C. for 18 hr. the minimum concentrations required to inhibit germination were noted and these are listed in Tables I and 2, Test A.

Table 3. Fumigant antifungal activities of certain ,&(thiocarbamoylthio)-aldehydes

Code no.

13 19 21 22 24 25 27 52

25 I

and -ketones and ~,3-dihydro-z-thi0-1,3-thiazines (Atmospheric concentration pg./ml. necessary to inhibit fungal growth.)

G. cingu- V . albo- A. brassi- M . fructi- A. niger lata atrum B. cinerea cicola

I 0 0 I 0 0 I 0 0 I 0 0 I 0 0 I 0 0 I 0 0 I 0

I I

I00 I I 0 0 I 0 0

> I 0 0 > I 0 0

I 0 0 > I00 I I00

I 0 > I 0 0 I > I 0 0 I I 0 0 I I 0 0 I I 0 0

I 0 0 > I 0 0

100 > I 0 0 I 0 0 I 0 0 I 0 0 > I 0 0

> I 0 0 > I 0 0 I 0 0 I00 I 0 0 I 0 0 1 0 0 > I 0 0

> I 0 0 > I 0 0 I 0 1 0 1 I 0' I 0 1

c. cucu- merinum

I

I 0 I00 I 0

I

I I 0 0 I 0 0

0' I

Fumigant test Preliminary agar-plate tests with these compounds had indicated that some of them

possessed fumigant antifungal activity. A simple test was conducted to eliminate inactive compounds. A solution of the compound (0.2 ml.) at 2000 ,ug./ml. in acetone was pipetted on to a strip of filter-paper and, after evaporation of the solvent, this was inserted into a culture tube containing a potato-dextrose agar slope inoculated with the spores of Botrytis cinerea. Under these conditions, complete vaporization would yield approximately z5,uglrnl. of air. Eight of the compounds were found to show some fumigant fungicidal activity, and these were examined further. Acetone solutions were pipetted on to filter papers and the solvent allowed to evaporate. Quantities used were such as to provide I , 10 and Ioo,ug./ml. of the air-space in inverted Petri dishes containing potato-dextrose agar. The plates, previously inoculated with the spores of Aspergillus niger, Glomerella cingulata, Verticillium albo-atrum, Botrytis cinerea, Alternaria brassicicola, Monilia fructigena and Cladosporium cucumeri- num, were inverted over the lids bearing the treated papers and sealed by pouring molten white soft paraffin into the space between lid and bottom. A compound of known fumigant fungicidal activity, 2,~,~-trichlorophenoxythio-trichloromethane

I20 G. A. CARTER AND OTHERS

(No. 251; Fawcett, Spencer & Wain, 1958) at concentrations of 0'1, I and ~opg./ml. air was used as standard, and control plates containing papers which had received acetone only were included. After 48 hr. at 25'C. the concentrations which in- hibited the growth of the respective fungi were noted and the results are given in Table 3,

Tests for protectant action AU test plants were raised in a heated greenhouse in 7 cm. diameter plastic pots

containing John Innes Compost. Uniformly germinating broad bean seedlings (var. Green Windsor) were grown, two per pot, until they possessed two pairs of fully expanded leaflets. Seeds of dwarf beans (var. Canadian Wonder) were planted, three per pot, and subsequently thinned to two seedlings per pot. They were grown on until the first pair of simple leaves were fully expanded. Wheat seedlings (var. Eclipse), pre-germinated on filter-paper at 22' C. for 3 days under red light, were planted, five per pot, and grown until the first leaf was still erect and approximately 12 cm. long.

All plants were sprayed to run-off with known concentrations of the test chemicals in 60 yo aqueous acetone. When the solvent had evaporated the plants were inoculated as follows.

Broad beam. Three replicate pots per treatment were sprayed with a suspension containing approximately 20,000 conidia of Botrytis fabae per ml., washed from 10-day-old cultures and resuspended in 0.2% sucrose solution. After 48 hr. in a humidity chamber, lesion counts were made within a circle 1-8 cm. diameter, as near to the leaf apex as possible. The remaining three replicate pots per treatment were sprayed with a heavy suspension, in 0-2 % sucrose, of uredospores of Uromyces fabue, washed from infected plants. After 48 hr. in a humidity chamber the plants were returned to the greenhouse for 1-14 days until the uredosori were sufficiently developed to enable counts to be made.

Dwarf beans. Three replicate pots per treatment were inoculated with a heavy suspension of the uredospores of Uromyces phuseolicola washed from infected plants. Subsequent treatment was as for U. fubae above.

Wheat. Three replicate pots per treatment were inoculated in a 6 ft. tower, with the dry conidia of Erysiphe graminis, brushed from infected plants. After dispersal throughout the tower by fans the spores were allowed to settle overnight. The plants were then returned to the greenhouse, watered with nutrient solution and left for 8-10 days until the mildew pustules were sufficiently developed to allow counts to be made.

The figures in Table 4 represent the mean number of lesions, uredosori or pustules developing on the leaves of treated plants, expressed as a percentage of control.

C H E M I C A L

(i) Determination of water solubility Sufficient of the finely ground substance to form a saturated aqueous solution was

shaken with distilled water for 2 hr. in a thermostatically controlled water bath at 25' C. The solution was filtered and the first few ml. of the filtrate discarded. After

Investigations on fungicides. XI I21

Table 4. Protectant activity of /3-(thbcarbanwylthio)-aZdehydes and -ketones and 2,3-dihydro-z-thi0-1,3-thiazines

Host and pathogen (infection as percentage of control)

A ,

20

22

26

27

30

49

51

53

Captan

Dinocap

Broad bean Dwarf bean Concentration , A , U.phaseolicoL

B. fabae

4 43

104

< I

13 23

7 30 32

7

26 89

I11

22

I < I

28 72 22

30

7

74 80 36 36 60

33 35 51 33 35 46 28 24 35

13 5 1

63

I 0

I 0

- - -

U. fabae

3 45

1 0 9

13

81

13 15 I3 55

22

< I I 0

25 I 0 0

< I

< I 16 73

9 7

I

< I 0 - 81

28 36 *4

*33 56

21

4 33 52

15 17 60

2 12

17 - - -

6 13 -

I

5

6 4 6

-

-

3

I9 2

-

2 I 1

24 -

2 3

I5 0

0 - -

4 7

26 6

15 32

6 I5 22

2 I 0

I9 3 3

I8 - - -

Wheat E. graminis

24

49 23

36

21

69 39 6 0 67 86

13 34

12

78 46 56 66 98 29 46 47 30 40 47 66

57 68 16 32 67 16 33 85

36

10

35 41 - - -

0

1 11

Phytotoxicity observed. - Not tested.

I22 G. A. CARTER AND OTHERS

dilution by a known factor, the concentration of the substance was determined by measuring its absorption at a suitable wavelength using a SP 500 Unicam ultra- violet spectrophotometer.

(ii) Determination of partition coejicient (water/oiZ) Castor oil was selected for this study in view of its slight solubility in aqueous

alcohol, thus enabling an optically clear solution to be obtained. A saturated solution of the substance in water, or a suitable dilution of this, was

used. Aliquots (20.0, 15.0 and 10.0 ml.) (q) were taken and added to sufficient water (0.0, 5-0 and 10.0 ml.) in a separatory funnel to make the volume up to 20.0 ml. (VJ. Castor oil (5-0 ml.) (v3) was added and the mixture shaken vigorously for 9 hr. in a mechanical shaker. After shaking, the oil and aqueous phases were allowed to separate. A portion of the lower aqueous layer was removed and 5.0 ml. pipetted into absolute alcohol (20 ml.). The optical density of this solution (El) was measured at a suitable wavelength, using a SP5oo Unicam Spectrophotometer, against a blank alcoholic solution obtained using the above procedure starting with water (20 ml.) and castor oil (5.0 ml.). The optical density of the aqueous solution before partition (Ez) was recorded at the same wavelength after pipetting 5.0 ml. of this solution, or a suitable dilution thereof, into absolute alcohol (20 ml.). Assuming that each substance obeys the Beer-Lambert law, the partition coefficient (K) was calculated using equation (I). The results are summarized in Tables 5 and 6, each figure being the mean of the three determinations

Owing to the low water-solubility of certain of the substances studied, the method was not sufficiently sensitive to allow the partition coefficient to be determined over a wider range of concentrations ; the possibility of molecular association in aqueous solution therefore cannot be excluded. The agreement for the three separate determi- nations using different concentrations of the substance in the aqueous phase was usually within the limits f 5 %, whilst the mean deviation for the partition coefficients for twenty-six compounds was 3.8 yo. The periods allowed for shaking and equilibration were kept to a minimum, as certain compounds appeared to be unstable in bright daylight.

RESULTS A N D D I S C U S S I O N

Spore germination tests The results given in Table I show that most P-(thiocarbamoy1thio)-aldehydes

and -ketones with alkyl substituents only (1-13) were characterized by low fungi- toxicity. 2,3-Dihydro-z-thio-1,3-thiazines derived from these compounds (28-40), whilst also showing a low order of fungitoxicity, were considerably more active. Amongst the thiazines there was a slight reduction in fungitoxicity when the hydro- gen atom attached to nitrogen (28-35) was replaced by a methyl group (36-40), otherwise the presence and positions of methyl substituents were without effect.

The relationship between fungitoxicity and certain physicochemical properties, in

Investigations on fungicides. XI 123 particular water solubility and partition coefficient, has been discussed (Woodcock, 1961). The greater in aitro fungitoxicity of certain z,3-dihydro-z-thio-1,3-thiazines (28-40) in comparison with their parent /I-(thiocarbamoy1thio)-aldehydes and -ketones (1-13) prompted a comparison of their water and lipoid solubilities. The results obtained are shown in Tables 5 and 6.

Table 5 . Water solubilities (moles kl) and partition coejicient (water/oil) of p-( thiocarbamoy1thio)-aldehydes and -ketones Cformula I on p . I I 6)

R, = H R, = Me A A < , , ,

Code Water Partition Code Water Partition R1 R, R, R, no. solubility coefficient no. solubility coefficient

H H H H Me Me Me Me

Rl

H H H H Me Me Me Me

H H H I 6 . 4 0 ~ I O - ~ Me H H 2 6.13" I O - ~

H Me H 3 497x10-, Me Me H 4 1 2 7 x I O - ~

H H H 5 1 . 1 7 ~ 10-1

H Me H 6 4 4 8 x 1 0 - ~ Me Me H 7 6.68 x I O - ~

H Me Me 8 9 ~ 1 6 x 1 0 - ~

I '99X 10-l 9 4'95X 1 0 - 2 I 0 5'54X 10-2 I1

5'43 x 10-1

1 . 1 9 ~ I O - ~ 12

6.21 x 10-'

2'02 x 10-1 2 . 7 2 ~ 10-1 13

9.00 x I O - ~

4 9 9 x 10-2

2'77 x 10-2

1.20 x IO-,

- -

1'12 x 1 0 - 2

3.12 x 10-1

3.38 x 10-l

1'02 x 10-1

1'93 X 10-1

- -

4 4 2 x I O - ~

Table 6. Water solubilities (moles I.-') and partition coejicient (water/oil) of 2,3-dihydro-z-thio-1,3-thiaxines Cformula 111 on p . I 16)

R, = H RS = Me

Ra

H Me H Me H H Me H

R3

H H Me Me H Me Me Me

R d

H H H H H H H Me

7

Code no.

28 29 30 31 32 33 34 35

Water solubility

1'12 x 1 0 - 2

3.06 x I O - ~

1.06 x I O - ~

2.87 x I O - ~

9.28 x I O - ~ 2.88 x I O - ~ 9'13 x 10-* 2'94 x 1 0 - 9

Partition coefficient

5'33 x 1 0 - 2

2.70 x I O - ~

1.61 x I O - ~

2'42 x 1 0 - 2

1.23 x I O - ~

6.1 I x I O - ~

1'99 X IO-;

1'39 X 10-a

7

Code no.

36

38 37

39

40

Water Partition solubility coefficient

1.10 x IO-* 3.61 X IO-' 6.54 x I O - ~

1.58 x I O - ~

1'49 X 10-a 1'30 x I O - ~

7.54 x 10-* - - - - - - -

1.07 x I O - ~ 2.46 x IO-,

All the p-(thiocarbamoy1thio)-aldehydes and -ketones possessing alkyl substituents only are thought to exist in neutral solution as cyclic structures (11, see p. 116) and are, in effect, primary or secondary alcohols (Jansen & Mathes, 1955; Garraway, 1964a). The ketones (Table 5 ; 5-8, 13) were found to be rather more water-soluble than the aldehydes (1-4, 9-12), whilst amongst the aldehydes N-methyl substituted compounds (9-1 2) tended to be more water-soluble than their unsubstituted analogues (1-4). The partition coefficients (water/oil) appeared to vary in a similar way, the compounds falling into three distinct groups (Fig. I). Within each of these groups methyl substitution, in general, depressed water solubility and increased lipoid solubility. Most 2,3-dihydro-z-thio-1,3-thiazines (Table 6; 28-40) were less water- soluble than the parent aldehydes and ketones (1-1 3); methyl substitution, particularly at the 5-position, resulted in decreased water solubility. The partition coefficient (water/oil) appeared to depend solely on the degree of methyl substitution (Fig. 2) and indicated a ten-fold increase in lipoid solubility over that of the parent compounds.

124 G. A. CARTER AND OTHERS

The greater fungitoxicity of the thiazines (Table 2; 28-40) would seem thus to correlate with increased lipophilic properties. At the same time the differences in partition coefficients between these derivatives and their parent compounds (1-13) are not sufficiently large to suggest that this is the dominant factor in fungitoxicity. This view is supported to some extent by the poor correlation between fungitoxicity and the number and position of methyl groups. Fungitoxicity must be ascribed, therefore, to some fundamental structural difference in the two groups of compounds. Certain 2,3-dihydro-2-thio-1,3-thiazines carrying no, or only one, methyl substituent

Number of methyl groups Number of methyl groups

Fig. I Fig. 2

Fig. I . Relationship of partition coefficient (waterloil) to the degree of methyl substitution of B-(thiocarbamoy1thio)-aldehydes and ketones (see formula II onp. 116). Compounds: 0 1-4, x 5-8, + 9-13. Fig. 2. Relationship of partition coefficient (waterloii) to the degree of methyl substitution of 2,3-dihydro-z-thio-1,j-thiazines (see formula I11 on p. I 16).

(28, 30 and 32) were found to decompose in the presence of acid to form the parent aldehydes or ketones (I, 3 and 5 ) but they still showed greater activity than the latter compounds. It would seem, therefore, that the thiazines are able to penetrate the fungal membranes more easily. Alternatively, lack of activity by the p-(thiocarbamoyl- thio)-aldehydes and -ketones could be due either to the absence of a double bond (see formula I1 on p. I 16) or the presence of the hydroxyl group possibly affecting the orientation of the molecule in the biophase or at the site of action. It was hoped to obtain more information concerning the latter hypothesis by studying the effects of the positions of methyl substituents on fungitoxicity, but it would appear that these do not impart sufficient lipophilic character to counteract the effect of the polar hydroxyl grOUP.

The presence of a ,8-phenyl group in certain p-(thiocarbamoy1thio)-aldehydes and

Investigations on fungicides. XI 125

-ketones (14-17) failed to improve their fungitoxicity (Table I) but certain N-methyl substituted compounds with a P-phenyl group (19-21) exhibited considerable activity. Examination of the ultraviolet absorption spectra of this group of compounds revealed the three fungitoxic members to have a different type of spectrum in the presence of acids. I t was concluded (Garraway, 1964a) that these three compounds in acidic media exist mainly as the non-cyclic form (I) (see formulae on p. 116). Since physiological media are usually weakly acidic it appears probable that this property is of considerable importance in fungitoxicity. In alcohol solution the spectra of these three compounds also underwent changes with time. Whilst it is possible, theoretically, for them to decompose forming methyl isothiocyanate and P-thiolketones, the former being very fungitoxic and the latter having metal chelating properties (Tanaka & Yokoyama, 1960), the spectral changes could not be identified with such reactions. 2,3-Dihydro-2-thio-1,3-thiazines with a phenyl group at the 6-position (41-47) all

exhibited a low order of fungitoxicity (Table 2). A comparison of the water solubilities and partition coefficients of this group and the parent compounds (14-21) could not be made using the methods described and so the importance of these properties in fungitoxicity cannot be assessed. Certain thiazines (42 and 43) underwent decomposition in the presence of acid forming the parent ketones ( I S and 16) (Garraway, 1964b), but such chemical reactivity appears to be of little importance in relation to fungi- toxicity since the compounds are only weakly active. It is possible that they have an inherent toxicity comparable with that of other thiazines, but are rendered ineffective by their low water solubility.

,&(Thiocarbamoylthio)-ketones having a trichloromethyl group (22, 23) showed considerable fungitoxicity (Table I). Activity was further enhanced, although only slightly, by a phenyl group (24) or by N-methylation (25-27), whilst an a-methyl group resulted in diminished toxicity (23 and 26). The trichloromethyl group was less effective in promoting toxicity when introduced in the 2,3-dihydro-2-thio-1,3- thiazines (48-53)) although compounds having this substituent were generally more toxic than other thiazines investigated (Table 2).

All the P-(thiocarbamoy1thio)-ketones of this group are thought to exist in neutral solution as the cyclic form (11) (see formulae on p. 116) whilst N-methyl substituted compounds have a non-cyclic structure (I) in the presence of acids (Garraway, 1964~). Whilst this property might be of importance in the fungitoxicity of certain phenyl- substituted compounds (19 and 20) it appears that here the effects of the trichloro- methyl group are dominant presumably through increasing the lipophilicity of the molecule (Horsfall, 1956). It should be noted that this grouping is also present in such highly active fungicides as captan (Kittleson, 1952) and a number of other sulphur-containing compounds (Fawcett el al. 1958; Wain, Sob6tka & Spencer, 1963). Certain of these /3-(thiocarbamoy1thio)-ketones (22-24) tended to form 2, 3- dihydro-2-thio-1,3-thiazines (48-so), but since other derivatives (7, 8, I 5 and 16) also possessed this property, but were only slightly active, it would seem to be of little importance in fungitoxic action.

The low fungitoxicity of the trichloromethyl substituted 2,3-dihydro-2-thio-1,3- thiazines (48-53) shown in Table z would seem to indicate that the potential fungi-

I 26 G. A. CARTER AND OTHERS

toxicity of this particular type of thiazine ring is of a low order. Low fungitoxicity might also be ascribed to low water solubility although information relating to this and partition coefficients is lacking.

No conclusions regarding mode of action can be drawn from the slopes of the dosage-response curves (Tables I and 2, Test B) for either group of compounds. I n general, the antifungal spectra (Tables I and 2, Test A) reflected the results obtained with Botrytis cinerea (Test B) with only a few compounds showing specific fungitoxic action.

Fumigant test Of the eight compounds selected for fumigant tests, all, except 52, were quite active

(Table 3), although of a lower order than that of 2,~,~-trichlorophenoxythiotrichloro- methane (25 I). All the active compounds were P-(thiocarbamoy1thio)-ketones with maximum antifungal activity being realized in conjunction with a trichloromethyl substituent (22, 24, 25, 27). The effect of N-methylation was variable.

Protectant activity When tested against four plant diseases the selected compounds all showed pro-

tectant activity (Table 4), although certain compounds (e.g. 19) were less effective than might have been expected from the results of spore germination tests (Tables I and 2). In general, the powdery mildew (Etysiphegraminis) was the least affected and none of the compounds exhibited activity comparable to that of dinocap (2,4-dinitro- 6-( I-methylhepty1)-phenyl crotonate).

Against the rust diseases, particularly that of dwarf bean ( Uromyces phaseohcola), certain compounds (22, 24-27) were only slightly less effective than captan (N- trichloro-methylthio-~-cyclohexene-~,z-dicarboximide) (Table 4). Rust on broad bean (U. fabae) was controlled more effectively than chocolate spot (Botrytis fabae).

The compounds exerting greatest protectant action (Table 4) were, in general, /3-(thiocarbamoy1thio)-ketones, the thiazines being considerably less effective. Maxi- mum protectant activity was again realized only in the presence of a trichloromethyl substituent.

REFERENCES

BLUESTONE, H. (1960). Method of killing fungi comprising contacting said fungi with a 2-thiotetrahydrothiazine. U.S. Patent, 2,920,996, 12 Jan. 1960.

CARTER, G. A,, GARRAWAY, J. L., SPENCER, D. M. & WAIN, R. L. (1963). Investigations on fungicides. VI. The antifungal activity of certain dithiocarbamic and hydroxyformic acid derivatives. Ann. appl. Biol. 51, 135.

FAWCETT, C. H., SPENCER, D. M. & WAIN, R. L. (1958). Investigations on fungicides. IV. (Ary1oxythio)-trichloromethanes. Ann. appl. Biol. 46, 65 I .

GARRAWAY, J. L. (1964~) . Derivatives of dithiocarbamic acids. J. chem. SOC. (in the Press). GARRAWAY, J. L. (1964b). Cyclic derivatives of dithiocarbamic acids. J. chem. SOC. (in the

Press). HORSFALL, J. G. (1956). Principles of fungicidal action. Waltham, Mass. : Chronica

Botanica Co. HORSFALL, J. G. & RICH, S. (1951). Fungitoxicity of heterocyclic nitrogen compounds.

Contr. Boyce Thompson Inst. 16, 313. JANSEN, J. E. & MATHES, R. A. (1955). Some thiazinethiols and their intermediate compounds.

J. Amer. chem. SOC. 77, 2866.

Invest2ations on fungicides. XI 127 KITTLESON, A. R. (1952). A new class of fungicides. Science, 115, 84. LITCHFIELD, J. T. & FERTIG, J. W. (1941). On a graphic dosage effect curve. Bull. Johns

Hopkins Hosp. 69, 276. OWENS, R. G. (1959). Effects of elemental sulphur, dithiocarbamates and related fungicides

on organic acid metabolism of fungus spores. Dev. Industr. Microbiol. I, 187. SPENCER, D. M. (1962). Polystyrene microbeakers for spore germination tests. Plant Path. 11, 41.

TANAKA, H. & YOKOYAMA, A. (1960). Studies on the sulphur-containing chelating agents. IV. Structure of copper chelate of P-mercaptoketones. Chem. pharm. Bull. (Japan), 8, 1012.

WAIN, R. L., SOB~TKA, W. & SPENCER, D. M. (1963). Investigations on fungicides. VIII. The fungicidal properties of some thiocarbamoylthionitroparaffins. Ann. uppl. Biol. 51, 445.

WOODCOCK, D. (I 96 I). Structure-activity correlationships of some anti-fungal compounds. In Fungicides in Agriculture and Horticulture, S.C.I. Monograph no. 15, page 92.