Metabolism and metabolites of dithiocarbamates in the plant pathogenic fungus leptosphaeria maculans

Article abstract of DOI:10.1021/jf302038a

Synthetic compounds containing a dithiocarbamate group are known to have a variety of biological effects and applications including antifungal, herbicidal, and insecticidal application. Leptosphaeria maculans is a fungal pathogen of crucifers able to detoxify efficiently the only plant natural product containing a dithiocarbamate group, the phytoalexin brassinin. To evaluate the effects of dithiocarbamates on L. maculans, a number of structurally diverse S-methyl dithiocarbamates containing indolyl, biphenyl, and benzimidazolyl moieties were synthesized, and their antifungal activities and metabolism by L. maculans were investigated. All dithiocarbamates were transformed by L. maculans through hydrolysis to the corresponding amines, which were less antifungal than the parent compounds. Two dithiocarbonates were shown to be much less antifungal than the corresponding dithiocarbamates. Results of this investigation indicate that S-methyl dithiocarbamates are not useful inhibitors of L. maculans and that their rates of transformation by L. maculans did not correlate with the antifungal activity of the particular compound.

Full text of DOI:10.1021/jf302038a

Article
pubs.acs.org/JAFC
Metabolism and Metabolites of Dithiocarbamates in the Plant
Pathogenic Fungus Leptosphaeria maculans
M. Soledade C. Pedras* and Vijay K. Sarma-Mamillapalle
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada
S
* Supporting Information
ABSTRACT: Synthetic compounds containing a dithiocarbamate group are known to have a variety of biological effects and
applications including antifungal, herbicidal, and insecticidal application. Leptosphaeria maculans is a fungal pathogen of crucifers
able to detoxify efficiently the only plant natural product containing a dithiocarbamate group, the phytoalexin brassinin. To
evaluate the effects of dithiocarbamates on L. maculans, a number of structurally diverse S-methyl dithiocarbamates containing
indolyl, biphenyl, and benzimidazolyl moieties were synthesized, and their antifungal activities and metabolism by L. maculans
were investigated. All dithiocarbamates were transformed by L. maculans through hydrolysis to the corresponding amines, which
were less antifungal than the parent compounds. Two dithiocarbonates were shown to be much less antifungal than the
corresponding dithiocarbamates. Results of this investigation indicate that S-methyl dithiocarbamates are not useful inhibitors of
L. maculans and that their rates of transformation by L. maculans did not correlate with the antifungal activity of the particular
compound.
KEYWORDS: antifungal, Brassicaceae, biotransformation, brassinin, crucifer, detoxification, dithiocarbamate, Leptosphaeria maculans,
Phoma lingam, phytoalexin
INTRODUCTION
■
Crop protectants against microbial diseases have been used in
agriculture for more than a century. The early fungicides
included salts of copper and mercury and sulfur dust.1 The first
patent awarded to Tisdale and Williams in 1934 represents a
landmark related to the development of dithiocarbamates as
fungicides, which were until then used as vulcanization
accelerators in the rubber industry.1 Besides fungicidal activity,
dithiocarbamates are known to have a variety of biological
effects and applications that include herbicidal and insecticidal
activity, regulation of apoptosis, pro- and antioxidant effects,
and therapy of alcohol aversion.2 The discovery of the first
Figure 1. Chemical structures of phytoalexins from crucifers: brassinin
plant natural product containing a dithiocarbamate group,
brassinin (1), established a curious link to the use of
dithiocarbamates in crop protection. Brassinin (1) is a
phytoalexin produced by cruciferous species, mainly oilseeds
such as canola (Brassica napus L., Brassica rapa L., Brasssica
juncea L.) and rapeseed (B. napus, B. rapa, B. juncea) and
horticultural crops, most of which belong to Brassica species.3
Brassica crops are of enormous importance worldwide as major
sources of vegetable oils, food, feed, and fuel. In addition to
brassinin (1), brassicas produce phytoalexins such as cyclo-
brassinin (2) and brassilexin (3), which are biosynthetically
derived from brassinin (1) and camalexin (4, Figure 1).
Somewhat surprisingly, to date no other plant species have
been reported to produce metabolites containing dithiocarba-
mates.
(1), cyclobrassinin (2), brassilexin (3), and camalexin (4).
[Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual
stage Phoma lingam (Tode ex Fr.) Desm.], one of the major
pathogens of canola (B. napus L., B. rapa L.), is able to detoxify
brassinin (1) by converting it to indole-3-carboxaldehyde (6,
Figure 2).5 All other fungal species investigated so far
(Alternaria brassicicola, Leptosphaeria biglobosa, L. maculans
(isolates virulent on brown mustard, B. juncea L.) and Botrytis
cinerea) converted brassinin (1) to the corresponding amine 9,
followed by acetylation (Figure 2).3 The enzyme involved in
the transformation of brassinin (1) to aldehyde 6, brassinin
oxidase (BOLm), was purified to homogeneity and shown to be
inducible and substrate specific.5 Screening of more than 60
compounds for inhibition of BOLm showed that the
Although an important part of the defensive mechanisms of
plants involves phytoalexins, these metabolites can be counter-
acted by detoxifying enzymes produced by phytopathogenic
fungi.4 In this connection, brassinin (1) was investigated and
found to be detoxified by diverse fungal species, including host-
specific and nonspecific species. The blackleg fungus
Received: May 10, 2012
Revised: July 20, 2012
Accepted: July 23, 2012
Published: July 23, 2012
© 2012 American Chemical Society
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mates; that is, all hydrolytic transformations occurring in
cultures of L. maculans are detoxifications.
MATERIALS AND METHODS
■
Materials. Chemicals were purchased from Sigma-Aldrich Canada
Ltd., Oakville, ON, Canada; solvents were of HPLC grade and used as
such. Flash column chromatography (FCC) was carried out using
silica gel grade 60, mesh size 230−400 Å or WP C18 prepscale bulk
packing 275 Å (J. T. Baker, Phillipsburg, NJ, USA).
Instruments. Nuclear magnetic resonance (NMR) spectra were
1
recorded on Bruker 500 MHz Avance spectrometers, for H at 500.3
MHz and for 13C at 125.8 MHz; chemical shifts (δ) are reported in
parts per million (ppm) relative to TMS; spectra were calibrated using
the solvent peaks; spin coupling constants (J) are reported to the
nearest 0.5 Hz. Fourier transform infrared (FT-IR) data were recorded
on a Bio-Rad FTS-40 spectrometer, and spectra were measured by the
diffuse reflectance method on samples dispersed in KBr. MS [high-
resolution (HR), electron impact (EI)] were obtained on a VG 70 SE
mass spectrometer employing a solids probe.
HPLC analysis was carried out with Agilent high-performance liquid
chromatographs equipped with quaternary pump, automatic injector,
diode array detector (DAD, wavelength range 190−600 nm), and
degasser. For method A (for neutral extracts), the column used was a
150 mm × 4.6 i.d., 5 μm, Eclipse XDB-C18 (Agilent), having an in-line
filter, and mobile phase 50% H2O/50−100% MeOH, for 25.0 min,
linear gradient, at a flow rate of 0.75 mL/min. For method B (for
amines and basic extracts), the column used was a 100 mm × 3.0 i.d.,
3.5 μm, Zorbax ODS (with both solvents containing 0.01% n-
propanamine) 40% H2O/60−100% MeOH, for 10.0 min, linear
gradient, at a flow rate of 0.50 mL/min.
Figure 2. Biotransformation of brassinin (1) by plant pathogenic
fungi: (i) Leptosphaeria maculans (isolates virulent on canola); (ii)
Alternaria brassicicola, L. biglobosa, and L. maculans (isolates virulent
on brown mustard) and Botrytis cinerea.
Synthesis and Characterization of New Compounds. All
synthetic compounds were purified using flash column chromatog-
raphy (FCC) on silica gel; satisfactory spectroscopic data identical to
those previously reported were obtained for all reported compounds.
Organic extracts were dried over Na2SO4. Syntheses of brassinin11
isobrassinin (1),12 1-methoxyisobrassinin (13a),12 compounds 5,13
11,14 and 20,11 were carried out as reported in the respective
publications, and compounds 16, 18, and 21 were prepared as
reported below and summarized in Figure 3.
phytoalexins cyclobrassinin (2), brassilexin (3), and camalexin
(4) were competitive inhibitors.5 The best synthetic inhibitors
of BOLm developed to date possess a scaffold based on
camalexin6 or brassilexin (3).7 The enzymes involved in the
hydrolysis of brassinin (1), brassinin hydrolases (BHs), were
isolated from L. maculans (BHLm, from isolates virulent on
brown mustard) and from A. brassicicola (BHAb);8 both
hydrolases were substrate specific and were inhibited
competitively by the phytoalexin cyclobrassinin (2) but not
by camalexin.
1-Methylbrassinin (1a) was transformed both in cultures of
L. maculans and by BOLm to the corresponding aldehyde 6a at
a rate similar to that of brassinin (1). S-Methyl tryptamine
dithiocarbamate (5), a homologue of brassinin (1), was not a
substrate of BOLm, but in cultures of L. maculans
dithiocarbamate 5 was transformed via different reactions: (i)
oxidative cyclization to dithiocarbamate 7 (not transformed
further) and (ii) hydrolysis to tryptamine (8) (Figure 2).9
These results suggest that L. maculans produces in culture, in
addition to BOLm, other enzymes that catalyze the hydrolysis
of dithiocarbamates and the oxidation of the indole nucleus.
Previously, three benzyl dithiocarbamates were also hydrolyzed
by L. maculans to the corresponding amines, which were then
acetylated in culture.10 That is, except for brassinin (1), the
transformation of dithiocarbamates by L. maculans appears to
occur via hydrolysis to amines; in all examples, these
transformations were detoxification reactions.
S-Methyl 4-Biphenyl-4-methyl Dithiocarbamate (16). A solution
of NH2OH·HCl (757 mg, 11.0 mmol) and Na2CO3 (873 mg, 8.23
mmol) in water (4 mL) was added to a solution of 4-
biphenylcaboxaldehyde (15, 1000 mg, 5.48 mmol) in EtOH (10
mL) and heated to reflux for 30 min. The reaction mixture was
concentrated to one-third, diluted with water, and extracted with
EtOAc. The combined organic extract was concentrated to yield a
mixture of (E,Z)-4-biphenylcarboxaldoximes (1049 mg, 5.32 mmol) in
97% yield. NaBH4 (234 mg, 6.18 mmol) and NiCl2·6H2O (245 mg,
1.03 mmol) were added to a cooled solution of the oximes (202 mg,
1.03 mmol) in MeOH (8 mL), and the reaction mixture was stirred at
room temperature. After 15 min, the reaction mixture was diluted with
1% NH4OH (30 mL) and filtered. The filtrate was extracted with
EtOAc (3 × 40 mL), and the organic extract was combined, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
separated by FCC (SiO2; MeOH/CH2Cl2/NH4OH, 20:80:1, v/v/v)
to afford 4-biphenylmethanamime (24, 170 mg, 0.93 mmol) in 91%
yield. CS2 (68 μL, 1.13 mmol) and MeI (88 μL, 1.40 mmol) were
added to a solution of amine 24 (170 mg, 0.94 mmol) and
triethylamine (256 μL, 1.88 mmol) in pyridine (2 mL) and stirred
at room temperature. After 30 min, the reaction mixture was diluted
with toluene and concentrated under reduced pressure. The residue
was separated by FCC (SiO2, EtOAc/hexane, 20:80, v/v) to yield 16
(185 mg, 72%, 0.68 mmol): mp 84 °C; HPLC tR = 23.9 min (method
A); 1H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 8 Hz, 4H), 7.48 (dd, J
= 8, 8 Hz, 2H), 7.42−7.38 (m, 3H), 7.27 (br s, NH), 4.98 (d, J = 5 Hz,
2H), 2.69 (s, 3H); signals for a minor rotamer were found at δ 4.66
and 2.72; 13C NMR (125 MHz, CDCl3) δ 199.5, 141.3, 140.7, 135.4,
129.1, 128.9, 127.8, 127.7, 127.3, 51.1, 18.5; FT-IR (KBr) υmax (cm−1)
3339, 3224, 2919, 1486, 1328, 1088, 926, 761, 697; UV (HPLC,
Considering the broad use of dithiocarbamates in agriculture,
and the need to design inhibitors of brassinin degradation by L.
maculans, it was of interest to investigate the potential
metabolism of structurally diverse dithiocarbamates. Here we
report that indolyl, biphenyl, and benzimidazolyl dithiocarba-
mate transformations in L. maculans are mediated by
hydrolases, the products of which are amines. All amines are
less toxic to the fungus than the corresponding dithiocarba-
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3H); 13C NMR (125 MHz, CDCl3) δ 199.1, 145.5, 141.8, 133.0,
125.9, 125.3, 121.2, 115.8, 20.1; FT-IR (KBr) νmax (cm−1) 3072, 1511,
1446, 1350, 1277, 1221, 1182, 1048, 1018, 825, 742; UV (HPLC,
MeOH/H2O) λmax (nm) 210, 262, 315; HRMS-EI calcd 208.0129 for
C9H8N2S2 ([M]+), found 208.0127; MS (EI) m/z (% relative
intensity) 208 [M+] (100), 161 (64), 134 (27), 91 (100), 63 (8).
S-Methyl 3-(3-Indolyl)propyl Dithiocarbonate (21). A solution of
acid 10 (500 mg, 2.67 mmol) in EtOH and H2SO4 was heated to
reflux for 20 h, and the solvent was removed under vacuum. The
reaction mixture was diluted with water and extracted with EtOAc, the
combined organic extract was concentrated, and the crude reaction
mixture was separated by FCC (SiO2; EtOAc/hexane, 10:90, v/v) to
yield ethyl indolyl-3-propanoate (545 mg, 2.51 mmol) in 96%. LAH
(116 mg, 3.06 mmol) was added in portions to a cooled solution of
the ethyl ester (550 mg, 2.55 mmol) in dry THF (5 mL), and the
reaction mixture was stirred at room temperature for 40 min. The
reaction mixture was quenched with NaOH solution (1 M, 5 mL), and
the precipitate was vacuum filtered. The filtrate was extracted with
EtOAc, and the combined organic extract was concentrated using a
rotary evaporator under reduced pressure. The residue was separated
by FCC (SiO2; 30% EtOAc/hexane) to afford indolyl-3-propanol (420
mg, 2.40 mmol) as a pale yellow oil in 95% yield. A solution of indolyl-
3-propanol (30 mg, 0.17 mmol) in THF (0.5 mL) was added to a
suspension of NaH (6 mg, 0.21 mmol) in THF (2 mL) and stirred at
room temperature for 5 min. Next, CS2 (19 μL, 0.31 mmol) followed
by MeI (20 μL, 0.31 mmol) was added, and the reaction mixture was
stirred at room temperature. After 20 min, the reaction mixture was
diluted with ice-cold water and extracted with EtOAc; the combined
organic extract was concentrated under reduced pressure using a rotary
evaporator to yield a residue that was separated by FCC (SiO2;
EtOAc/hexane, 20:80, v/v) to afford S-methyl 3-(3-indolyl)propyl
dithiocarbonate (21, 33 mg, 0.12 mmol) as a yellow viscous material in
1
72% yield: HPLC tR = 21.4 min (method A); H NMR (500 MHz,
CDCl3) δ 7.94 (br s, NH), 7.64 (d, J = 8 Hz, 1H), 7.38 (d, J = 8 Hz,
1H), 7.25 (dd, J = 7.5, 7.5 Hz, 1H), 7.17 (dd, J = 7.5, 7.5 Hz, 1H), 7.01
(s, 1H), 4.69 (t, J = 6.5 Hz, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.60 (s, 3H),
2.25 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 216.1, 136.5, 127.4,
122.2, 121.7, 119.4, 118.9, 115.1, 111.3, 73.8, 28.7, 21.6, 19.1; FT-IR
(KBr) νmax (cm−1) 3414, 2921, 1456, 1223, 1056, 742; UV (HPLC,
MeOH/H2O) λmax (nm) 220, 278; HRMS-EI calcd 265.0595 for
C13H15NOS2 ([M]+), found 265.0587; MS (EI) m/z (% relative
intensity) 265 [M+] (33), 232 (52), 218 (32), 157 (52), 130 (100).
General Procedure for Acetylation of Amines 22−25. Acetic
anhydride (1.1 equiv) was added to a solution of each amine (1 equiv)
and pyridine (1.5 equiv) in CH2Cl2 (2 mL) at 0 °C and stirred at
room temperature, as described below. After complete conversion of
the starting material, the reaction mixture was diluted with toluene (5
mL) and concentrated using a rotary evaporator to afford crude
substituted acetamides that were separated by FCC (SiO2; EtOAc).
Nb-Acetyl-2-indolylmethanamine (22a). Acetylation of indolyl-2-
methanamine (22, 30 mg, 0.20 mmol) using the above procedure
afforded compound 22a (29 mg, 0.15 mmol) in 75% yield: mp 109
°C; HPLC tR = 6.2 min (method A); 1H NMR (500 MHz, CDCl3) δ
9.05 (br s, NH), 7.57 (d, J = 8 Hz, 1H), 7.33 (d, J = 8 Hz, 1H), 7.17
(dd, J = 7.5, 7.5 Hz, 1H), 7.09 (dd, J = 7.5, 7.5 Hz, 1H), 6.32 (s, 1H),
6.18 (br s, NH), 4.45 (d, J = 6 Hz, 2H), 2.00 (s, 3H);13C NMR (125
MHz, CDCl3) δ 171.9, 136.6, 127.8, 122.2, 120.5, 119.9, 111.3, 100.7,
37.6, 23.2; FT-IR (KBr) νmax (cm−1) 3392, 3269, 3060, 1651, 1531,
1456, 1423, 1288, 749; UV (HPLC, MeOH/H2O) λmax (nm) 219,
Figure 3. Syntheses of dithiocarbamates and dithiocarbonates.
Reagents and reaction conditions: (a) (i) EtOH, H2SO4, reflux,
96%; (ii) DiBAl-H, toluene, −78 °C, 90%; (iii) NH2OH·HCl,
Na2CO3, EtOH/H2O, 97%; (iv) NaBH4, NiCl2·6H2O, MeOH; (v)
Pyr, Et3N, CS2, CH3I, 43%; (b) (i) EtOH, H2SO4, reflux, 87%; (ii)
LAH, THF, 0 °C; (iii) MnO2, DCM, 80%; (iv) NH2OH·HCl,
Na2CO3, EtOH/H2O, 94%; (v) NaBH4, NiCl2·6H2O, MeOH; (vi)
Pyr, Et3N, CS2, CH3I, 44%; (c) (i) t-BuLi, THF, −78 °C; (ii) DMF,
86%; (iii) NH2OH·HCl, Na2CO3, EtOH/H2O, 93%; (iv)
NaBH3(CN), TiCl3, NH4OAc, MeOH; (v) Pyr, Et3N, CS2, CH3I,
51%; (d) (i) NH2OH·HCl, Na2CO3, EtOH/H2O, 97%; (ii) NaBH4,
NiCl2·6H2O, MeOH, 91%; (iii) Pyr, Et3N, CS2, CH3I, 72%; (e) (i)
NaH, THF, 0 °C; (ii) CS2, CH3I, 72%; (f) (i) NaH, THF, CS2; CH3I,
90%; (g) (i) EtOH, H2SO4, reflux, 96%; (ii) LAH, THF, 0 °C, 95%;
(iii) NaH, THF, 0 °C; (iv) CS2, CH3I, 73%.
MeOH/H2O) λmax (nm) 204, 260; HRMS-EI calcd 273.0646 for
C15H15NS2 ([M]+), found 273.0646; MS (EI) m/z (% relative
intensity) 273 [M+] (16), 225 (20), 167 (100).
S-Methyl Benzimidazolyl Dithiocarbamate (18). A solution of
benzimidazole (17, 100 mg, 0.85 mmol) in THF (1 mL) was added to
a stirred suspension of NaH (26 mg, 1.1 mmol) in THF (1 mL). After
5 min, CS2 (61 μL, 1.02 mmol) and MeI (80 μL, 1.27 mmol) were
added to the reaction mixture at room temperature, which was further
stirred for 20 min; the reaction mixture was diluted with ice-cold water
and extracted with EtOAc. The combined organic extract was
concentrated under reduced pressure to yield a residue that was
separated by FCC (SiO2; EtOAc/hexane, 20:80, v/v) to yield S-methyl
benzimidazolyl dithiocarbamate (18, 126 mg, 0.61 mmol) as a yellow
solid in 72% yield: mp 63 °C (lit. 58 °C,15); HPLC tR = 11.2 min
(method A); 1H NMR (500 MHz, CDCl3) δ 8.88 (s, 1H), 8.54 (d, J =
7.5 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.44−7.38 (m, 2H), 2.85 (s,
+
272; HRMS-EI m/z measured 188.0947 ([M] , calcd 188.0950 for
C11H12N2O); MS (EI) m/z (% relative intensity) 188 [M+] (100), 145
(46), 130 (40), 118 (51).
Nb-Acetyl-1-methoxy-2-indolylmethanamine (23a). Acetylation
of 1-methoxy-2-indolylmethanamine (23, 209 mg, 1.19 mmol) using
the general procedure afforded compound 23a (223 mg, 1.02 mmol)
in 84% yield as a pale yellow semisolid: HPLC tR = 8.9 min (method
A); 1H NMR (500 MHz, CDCl3) δ 7.52 (d, J = 8 Hz, 1H), 7.39 (d, J =
8 Hz, 1H), 7.24 (dd, J = 8, 8 Hz, 1H), 7.11 (dd, J = 8, 8 Hz, 1H), 6.32
(br s, NH), 6.24 (s, 1H), 4.58 (d, J = 6 Hz, 2 H), 4.04 (s, 3H), 1.98 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 133.3, 132.7, 123.7,
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122.5, 121.0, 120.5, 108.2, 97.4, 65.5, 34.7, 23.2; FT-IR (KBr) νmax
(cm−1) 3275, 3060, 2940, 1655, 1541, 1452, 1288, 971, 744; UV
(HPLC, MeOH/H2O) λmax (nm) 220, 270; HRMS-EI m/z measured
Table 1. Antifungal Activities of Brassinin,
Dithiocarbamates, and Dithiocarbonates against
Leptosphaeria maculans
+
218.1050 ([M] , calcd 218.1055 for C12H14N2O2); MS (EI) m/z (%
relative intensity) 218 [M+] (28), 187 (44), 169 (27), 145 (100), 129
(12), 118 (70), 89 (14).
a
inhibition SD (%)
compound
brassinin (1)
0.50 mM
0.20 mM
0.10 mM
Nb-Acetyl-3-(3-indolyl)propanamine (24a). Acetylation of 3-(3-
indolyl)propanamine (24, 150 mg, 0.867 mmol) using the above
procedure afforded compound 24a (138 mg, 0.64 mmol) in 74% yield
as yellowish crystals: mp 93−95 °C (lit.16 97−99 °C); HPLC tR = 21.4
55 3 d
ci b
21 3 fg
71 3 c
10 4 def
43 2 b
S-methyl tryptamine
dithiocarbamate (5)
1
S-methyl 3-(3-indolyl)propyl
dithiocarbamate (11)
67 3 c
46 5 d
21 5 c
min (method A); H NMR (500 MHz, CDCl3) δ 8.54 (br s, NH),
7.58 (d, J = 8 Hz, 1H), 7.34 (d, J = 8 Hz, 1H), 7.20 (dd, J = 8, 7 Hz,
1H), 7.12 (dd, J = 8, 7 Hz, 1H), 6.90 (s, 1H), 5.86 (br s, NH), 3.28
(m, 2H), 2.77 (t, J = 7 Hz, 2H), 1.91 (s, 3H), 1.89 (quint, J = 7 Hz,
2H); 13C NMR (125 MHz, CDCl3) δ 170.6, 136.6, 127.4, 121.9,
121.8, 119.1, 118.8, 115.2, 111.5, 39.7, 29.8, 23.3, 22.7; FT-IR (KBr)
νmax (cm−1) 3403, 3283, 2930, 1652, 1550, 1456, 743; UV (HPLC,
MeOH/H2O) λmax (nm) 221, 282; HRMS-EI m/z measured 216.1266
isobrassinin (13)
43 4 e
34 2 f
46 4 e
22 4 f
15 3 g
32 4 e
13 3 de
1-methoxyisobrassinin (13a)
7
3 e,f
S-methyl 4-biphenyl-4-methyl
dithiocarbamate (16)
14 4 d
S-methyl benzimidazolyl
dithiocarbamate (18)
ci b
85 3 b
15 2 g
18 2 fg
43 5 b
+
S-methyl 3-(3-indolyl)propyl
dithiocarbonate (20)
32 4 f
31 2 f
7
5
3 ef
3 f
([M] , calcd 216.1263 for C13H16N2O); MS (EI) m/z (% relative
intensity) 216 [M+] (39), 157 (35), 144 (37), 130 (100).
S-methyl tryptophol
dithiocarbonate (21)
N-Acetyl-4-biphenylmethanamine (25a). Acetylation of 4-biphe-
nylmethanamine (225 mg, 1.24 mmol) using the above procedure
afforded compound 25a (25, 251 mg, 1.11 mmol) in 90% yield:
darkening 158 °C; mp 184 °C (lit.17 180−182 °C); HPLC tR = 18.3
a
The percentage of inhibition was calculated using the following
formula: % inhibition = 100 − [(diameter of mycelia on amended/
diameter of mycelia on control) × 100]; values are averages of three
independent experiments conducted in triplicate; ci = complete
inhibition. For statistical analysis, one-way ANOVA tests were
performed followed by Tukey’s test with adjusted α set at 0.05; n =
6; entries with the same letters (b−f) within each column are not
significantly different (P < 0.05).
1
min (method A); H NMR (500 MHz, CDCl3) δ 7.64 (d, J = 8 Hz,
2H), 7.61 (d, J = 8 Hz, 2H), 7.46 (dd, J = 7, 8 Hz, 2H), 7.36 (m, 3H),
6.85 (br s, NH), 4.36 (d, J = 6 Hz, 2H), 1.92 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 171.0, 142.0, 140.9, 140.3, 130.3, 129.3, 128.7, 128.3,
128.2, 43.6, 23.4; FT-IR (KBr) νmax (cm−1) 3292, 3091, 1638, 1554,
1443, 1373, 1294, 1094, 756; UV (HPLC, MeOH/H2O) λmax (nm)
+
206, 252; HRMS-EI m/z measured 225.1148 ([M] , calcd 225.1154
for C15H15NO); MS (EI) m/z (% relative intensity) 225 [M+] (100),
182 (66), 167 (34), 106 (20).
Antifungal Bioassays and Metabolism of Dithiocarbamates
and Dithiocarbonates by Leptosphaeria maculans. The
antifungal activity of compounds was determined using a mycelial
radial growth bioassay.11 In brief, isolates of L. maculans (isolate BJ-
125 or UAMH-9410) were grown on V8 agar plates. Potato dextrose
agar (PDA) sterile tissue culture 6-well plates (33 mm diameter)
containing 0.50, 0.20, and 0.10 mM of each test compound (dissolved
in CH3CN) were used; control plates contained only 1% CH3CN in
PDA. Wells were inoculated with 4 mm mycelial plugs, cut from 7-day-
old V8 agar plates of L. maculans, placed upside down on the center of
each plate and incubated under constant light for 5 days. Mycelial
growth (diameter) in each treatment was measured, and the percent
inhibition was calculated according to the following equation: %
inhibition = 100 − [(growth diameter on amended medium/growth
diameter in control medium) × 100]. All bioassay experiments were
carried out in triplicate, at least twice.
For investigation of metabolic transformations, Erlenmeyer flasks
(150 mL) containing minimal medium10 (50 mL) were inoculated
with spores of L. maculans (isolate BJ-125, 106/mL) and incubated at
23 °C on a shaker at 120 rpm under constant light. After 2 days,
compounds dissolved in CH3CN were added to the cultures (final
concn 0.10 mM) as well as to noninoculated medium (stability
control). Samples (2 or 5 mL) were withdrawn immediately and at
intervals up to 72 h and were either frozen or immediately extracted
with EtOAc, the organic extracts were concentrated, and the residue
was dissolved in CH3CN and analyzed by HPLC using method A. The
extracted broth was basified by adding NH4OH (pH ≥9) and
extracted with CHCl3/MeOH (95:5). The concentrated extract was
analyzed by HPLC using method B. Calibration curves of each
compound were built for quantitative analysis using HPLC-DAD.
Figure 4. Progress curves for transformation of brassinin (1) and
dithiocarbamates 5, 11, 13, 13a, 16, and 18 by Leptosphaeria maculans.
Concentrations were determined by HPLC using calibration curves;
each point is the average of at least three independent experiments
standard deviations.
of dithiocarbonates 20 and 21 were carried out by treatment of
the corresponding alcohols with carbon disulfide, followed by
methylation with methyl iodide, as summarized in Figure 3.
Antifungal Activity. The antifungal activity of each
compound against L. maculans was determined, and results
are summarized in Table 1. S-Methyl benzimidazolyl
dithiocarbamate (18) and S-methyl tryptamine dithiocarbamate
(5) were the most potent mycelial growth inhibitors among all
compounds tested, displaying significantly higher inhibitory
activity than brassinin (1). The inhibitory activity of
dithiocarbonates 20 and 21 was similar to that of 1-
methoxyisobrassinin (13a) and the lowest among all
RESULTS AND DISCUSSION
■
Chemical Synthesis. Syntheses of brassinin (1) and
dithiocarbamates 5, 11, 13, 13a, and 16 were accomplished
by treatment of the corresponding amines with CS2, followed
by methylation with MeI as summarized in Figure 3. Syntheses
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compounds were hydrolyzed by the fungus in the medium,
albeit at different rates (all compounds were stable in medium
alone, giving comparable recoveries). Progress curves for the
transformation of each dithiocarbamate (Figure 4) indicated
that tryptamine dithiocarbamate (5), 3-(3-indolyl)propyl
dithiocarbamate (11), and 1-methoxyisobrassinin (13a) were
metabolized substantially more slowly than brassinin (1),
whereas isobrassinin (13), 4-biphenyl-4-methyl dithiocarba-
mate (16), and benzimidazolyl dithiocarbamate (18) were
metabolized at rates similar to that of brassinin (1) (Figures 4
and 5). Dithiocarbonate 20 was metabolized to the
corresponding alcohol 26 at a somewhat slower rate than 21
(24 vs 48 h, respectively), which was metabolized to 27. Except
for brassinin (1), which yielded aldehyde 6, all dithiocarba-
mates were hydrolyzed to the corresponding amines; amines
22−25 were further transformed to the corresponding acetyl
derivatives 22a−25a, whereas benzimidazole (17) was not
transformed further and remained in culture for the duration of
the experiment (72 h) (Figures 4 and 5). In addition, as
previously shown,9 dithiocarbamate 5 was mainly transformed
through two pathways that yielded dithiocarbamate 7 and
tryptamine (8). Dithiocarbamate 7 remained in culture for the
duration of the experiment, thus appearing to be the only
dithiocarbamate that L. maculans is unable to transform. These
studies, that is, products of each transformation, amounts, and
reaction times, are summarized in Table 2.
To establish if the transformations of dithiocarbamates 5, 11,
13, 13a, 16, and 18 and dithiocarbonates 20 and 21 by L.
maculans were detoxifications, the products of each trans-
formation, that is, amines 22, 23, and 25, acetyl amines 22a,
23a, and 25a, benzimidazole (17), and alcohols 26 and 27 were
assayed for mycelial growth inhibition of L. maculans (Table 3).
All of these products were less inhibitory than the parent
compounds; 4-biphenyl-4-methanamine (24) was not soluble
in the agar medium and thus could not be evaluated.
Considering the substantially higher inhibitory activity of
dithiocarbamates 5, 11, 13, 13a, 16, and 18 than those of
their biotransformation products, it is concluded that L.
maculans is able to detoxify these dithiocarbamates to less
toxic products. On the other hand, the inhibitory activities of
the products of transformation of dithiocarbonates 20 and 21
were similar to those of the parent compounds.
Figure 5. Biotransformation reactions of synthetic dithiocarbamates
11, 13, 13a, 16, and 18 and dithiocarbonates 20 and 21 by
Leptosphaeria maculans (isolates virulent on canola).
compounds; however, dithiocarbamate 11 was also more active
than 1.
Metabolism of Dithiocarbamates 5, 11, 13, 13a, 16,
and 18 and Dithiocarbonates 20 and 21. To determine if
synthetic dithiocarbamates 5, 11, 13, 13a, 16, and 18 and
dithiocarbonates 20 and 21 were metabolized by L. maculans,
each compound dissolved in acetonitrile was added to cultures
and to noninoculated media (stability control) to obtain a final
concentration of 0.10 mM. HPLC analysis of neutral and basic
extracts after different incubation times revealed that all
Table 2. Products of Metabolism of Dithiocarbamates and Dithiocarbonates in Cultures of Leptosphaeria maculans
recovery after
a
compound
brassinin (1)
24 h (%)
major products of metabolism, molar % (time)
<5
55
indole-3-carboxaldehyde (6), 40% (15 h)
S-methyl tryptamine
dithiocarbamate (5)
methyl 3a-hydroxy-3,3a,8,8a-tetrahydropyrrolo[2,3-b]indol-1(2H)-yl carbodithioate (7), 38%; tryptamine
(8), <5%; Nb-acetyltryptamine (8a), 20% (72 h)
S-methyl 3-(3-indolyl)propyl
dithiocarbamate (11)
37
3-(3-indolyl)propanamine (25), <5%; Nb-acetyl-3-(3-indolyl)propanamine (25a), 6% (24 h)
isobrassinin (13)
<5
47
2-indolylmethanamine (22), <5%; Nb-acetyl-2-indolylmethanamine (22a), 30% (48 h)
1-methoxyisobrassinin (13a)
1-methoxy-2-indolylmethanamine (23), <5%; Nb-acetyl-1-methoxy-2-indolylmethanamine (23a), 37%
(48 h)
S-methyl 4-biphenyl
dithiocarbamate (16)
<5
10
4-biphenylmethanamine (24), <5%; N-acetyl-4-biphenylmethanamine (24a), 21% (72 h)
benzimidazole (17), 62% (24 h)
S-methyl benzimidazolyl
dithiocarbamate (18)
S-tryptophol dithiocarbonate (20)
15
nd
tryptophol (26), 32% (24 h)
S-methyl 3-(3-indolyl)propyl
dithiocarbonate (21)
3-(3-indolyl)-1-propanol (27), 60% (24 h)
a
The percentage of each product was determined using calibration curves and is the average of at least three experiments conducted in triplicate.
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Table 3. Antifungal Activity of Amines, Acetylamines, Benzimidazole, and Alcohols against Leptosphaeria maculans
a
inhibition SD (%)
compound
0.50 mM
0.20 mM
0.10 mM
tryptamine (8)
28 3 bc
15 3 e
35 3 b
33 2 b
33 4 b
19 3 de
15 2 e
30 5 bc
27 5 bc
24 3 cd
29 5 bc
18 3 de
15 5 bcd
12 4 bc
Nb-acetyltryptamine (8a)
7
3 e
ni d
11 3 b
ni d
benzimidazole (17)
20 2 b
19 4 b
18 3 bc
2-indolylmethanamine (22)
Nb-acetyl-2-indolylmethanamine (22a)
1-methoxy-2-indolylmethanamine (23)
Nb-acetyl-1-methoxy-2-indolylmethanamine (23a)
N-acetyl-4-biphenylmethanamine (24a)
3-indolylpropanamine (25)
7
ni d
4
2 bcd
8
4 de
11 3 cde
17 4 bc
3 cd
4 bc
9
14 4bcd
13 4 bcde
16 4 bcd
14 3 bcde
ni d
Nb-acetyl-3-(3-indolyl)propanamine (25a)
tryptophol (26)
10 3 bc
ni d
3-(3-indolyl)-1-propanol (27)
7
2 cd
a
The percentage of inhibition was calculated using the following formula: % inhibition = 100 − [(growth on amended/growth in control) × 100]; ni
= no inhibition. For statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted α set at 0.05; n = 6; entries
with the same letters (b−f) within each column are not significantly different (P < 0.05).
In summary, the potential metabolism of dithiocarbamates
containing indolyl, biphenyl, and benzimidazolyl moieties by L.
maculans and their antifungal activities were investigated. All
dithiocarbamates were transformed via hydrolysis to the
corresponding amines, unlike brassinin (1), which was
transformed to indole-3-carboxaldehyde (6). That is, whereas
the transformation of brassinin (1) was catalyzed by an
oxidase,5,6 the transformations of dithiocarbamates 5, 11, 13,
13a, 16, and 18, evaluated for the first time in this work, were
likely catalyzed by hydrolases. It is likely that these hydrolases
are “house-keeping” enzymes used in the metabolism of
xenobiotics. The products of these hydrolyses (amines 8, 22,
23, and 25) were less toxic to the fungus than the
corresponding dithiocarbamates; that is, these hydrolase-
mediated transformations occurring in L. maculans are
detoxifications. It is worth noting that the rates of trans-
formation of dithiocarbamates 5, 11, 13, 13a, 16, and 18 did
not correlate with their inhibitory activities. For example, S-
methyl tryptamine dithiocarbamate (5), one of the most
inhibitory dithiocarbamates tested, was transformed at a rate
similar to that measured for 1-methoxyisobrassinin (13a), one
of the least inhibitory compounds.
(Discovery Grant to M.S.C.P.), the Canada Research Chairs
program, Canada Foundation for Innovation, the Saskatchewan
Ggovernment, and the University of Saskatchewan (graduate
assistantship to V.K.S.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the technical assistance of K. Brown (NMR)
and K. Thoms (MS) from the Department of Chemistry.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Progress curves for transformation of dithiocarbonates 20 and
21 by Leptosphaeria maculans and 1H and 13C NMR spectra of
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: s.pedras@usask.ca. Phone: 1 (306) 966-4772. Fax: 1
(306) 966-4730.
■
Funding
(10) Pedras, M. S. C.; Khan, A., Q.; Smith, K. C.; Stettner, S. L.
Preparation, biotransformation, and antifungal activity of methyl
benzyldithiocarbamates. Can. J. Chem. 1997, 75, 825−828.
Financial support for the authors’ work was obtained from the
Natural Sciences and Engineering Research Council of Canada
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