Cyclobrassinin Detoxification by Mimicry
J. Agric. Food Chem., Vol. 47, No. 3, 1999 1199
FTIR, UV) of the purified metabolites and confirmed by
synthesis, as described below.
100), 146 (15), 142 (16); CIMS, m/ z (%) 175 (M+ + 1, 100),
145 (4); HREIMS, m/ z calcd for C9H6N2S 174.0252, found
174.0253.
Syn th esis of P h ytoa lexin s a n d Th eir Meta bolites.
Cyclobrassinin (Takasugi et al., 1988) and dioxibrassinin
(Monde et al., 1991) were synthesized as previously described.
Brassicanal A (2). A solution of diazomethane (excess) in
Et2O was added to a solution of aldehyde 7 (20.3 mg, 0.11
mmol) in Et2O (1 mL). The reaction mixture was kept at room
temperature for 4 h. The solvent was then allowed to evaporate
overnight in the fumehood. This afforded 21.9 mg of brassi-
canal A (100% yield): HPLC tr ) 10.2 min. Remaining data
were identical to those previously reported (Pedras and Khan,
1996; Monde et al., 1990).
2-Mercaptoindole-3-carboxaldehyde oxime (13). To a solution
of aldehyde 7 (100 mg, 0.56 mmol) in ethanol (2 mL) was added
1 M NH2OH (1 mL, 1.1 mmol). The mixture was refluxed for
1 h and diluted with water (10 mL), and the resulting
precipitate was filtered and dried to yield 47.6 mg of oxime
13. The filtrate was extracted with EtOAc (2 × 20 mL). The
combined EtOAc extracts were dried over Na2SO4, and the
solvent was evaporated under reduced pressure to furnish
another 65.8 mg of oxime 13. Overall yield of the oxime was
1
113.4 mg (100%): HPLC tr ) 17.4 min; H NMR (300 MHz,
2-Mercaptoindole-3-carboxaldehyde (7). NaH (60%, 745 mg,
0.02 mol washed with hexane) was added slowly to a solution
of 2-indolinethione [250.0 mg, 1.7 mmol, prepared according
to the method of Hino et al. (1969)] in HCOOEt (5 mL, 0.06
mol), and the reaction mixture was kept at room temperature.
After 12 h, the reaction mixture was diluted with water (10
mL, dropwise addition with cooling), the mixture was acidified
with 2.5 M HCl, and the precipitate formed was filtered and
was washed with water. The precipitate was dried to yield
106.0 mg of aldehyde 7. The combined washings were acidified
with 2.5 M HCl and combined with the filtrate. The filtrate
was then extracted with EtOAc (2 × 120 mL), the combined
extracts were dried over Na2SO4, and the solvent was evapo-
rated under reduced pressure to yield 168 mg of 7; total yield
of aldehyde 7 was 274 mg (92%): HPLC tr ) 18.8 min; 1H NMR
(300 MHz, CD3OD) δ 9.37 (s, 1H), 8.15 (d, J ) 8.0 Hz, 1H),
7.48 (d, J ) 8.0 Hz, 1H), 7.37 (dd J ) 8.0, 8.0 Hz, 1H), 7.27
(dd, J ) 8.0, 8.0 Hz, 1H); 1H NMR (300 MHz, CD3CN) δ 10.58
(br s, 1H, D2O exchangeable), 9.65 (s, 1H), 8.12 (d, J ) 8.0
Hz, 1H), 7.52 (d, J ) 8.0 Hz, 1H), 7.37 (dd, J ) 8.0, 8.0 Hz,
1H), 7.29 (dd, J ) 8.0, 8.0 Hz, 1H); 13C NMR (75.5 MHz, CD3-
OD) δ 187.1, 139.1, 136.1, 126.9, 126.6, 124.5, 122.7, 122.3,
113.2; FTIR (cm-1) 3229, 1621, 1454, 1247, 1216, 742; EIMS,
m/ z (%) 177 (M+, 100), 149 (18), 148 (24), 121 (15); CIMS, m/ z
(%) 178 (M+ + 1, 19), 164 (32), 150 (24), 146 (100), 132 (50);
HREIMS, m/ z calcd for C9H7NOS 177.0248, found 177.0249.
3-Methylenaminoindole-2-thione (11). Titanium trichloride
(103 mg, 0.66 mmol) was added in small portions to a solution
of oxime 13 (64 mg, 0.33 mmol), NaBH3CN (63 mg, 1 mmol),
and NH4OAc (32 mg, 0.4 mmol) in MeOH (3 mL). After stirring
for 30 min at room temperature, the reaction mixture was
diluted with water (20 mL) and extracted with Et2O (3 × 20
mL). The combined Et2O extracts were washed with water (120
mL) and dried over Na2SO4, and the solvent was evaporated
under reduced pressure to yield 50.1 mg of thione 11 (85%):
HPLC tr ) 7.2 min; 1H NMR (500 MHz, CD2Cl2) δ 11.13 (br s,
1H, D2O exchangeable), 8.98 (br s, 1H, D2O exchangeable), 8.15
(dd, J ) 14.8, 8.2 Hz, 1H), 7.41 (d, J ) 7.5 Hz, 1H), 7.11 (m,
CD3CN) δ 9.92 (br s, 1H, D2O exchangeable), 8.52 (s, 1H, D2O
exchangeable), 8.04 (d, J ) 8.0 Hz, 1H), 7.95 (s, 1H), 7.42 (d,
J ) 8.0 Hz, 1H), 7.31 (dd, J ) 8.0, 7.0 Hz, 1H), 7.18 (dd, J )
8.0 Hz, 7.0 Hz, 1H); 13C NMR (75.5 MHz, CD3CN) δ 145.2 (d),
138.9 (s), 131.1 (s), 126.0 (d), 125.7 (s), 123.5 (d), 122.4 (d),
117.2 (s), 112.6 (d); FTIR (cm-1) 3383, 3323, 1618, 1405, 1340,
844, 744; EIMS, m/z (%) 174 (M+ - 18, 100), 149 (51);
HREIMS, m/z calcd for C9H6N2S (C9H8N2OS - H2O) 174.0252,
found 174.0251.
1
5-Hydroxybrassicanal A (8): HPLC tr ) 5.0 min; H NMR
(500 MHz, CD3CN) δ 10.06 (s, 1H), 9.96 (br s, 1H, D2O
exchangeable), 7.45 (d, J ) 2.5 Hz, 1H), 7.26 (d, J ) 9.0 Hz,
1H), 6.74 (dd, J ) 9.0, 2.5 Hz, 1H), 6.69 (s, 1H, D2O
exchangeable), 2.61 (s, 3H); 13C NMR (125.77 MHz, CD3OD)
δ 185.7, 154.9, 148.1, 133.0, 128.3, 117.3, 114.1, 112.7, 106.0,
17.0; FTIR (cm-1) 3171, 1612, 1585, 1444, 1365, 1224, 1083,
861; HRMS, m/ z measured 207.0353 (207.0354 calcd for C10H9-
NO2S); EIMS, m/ z (intensity) 207 (M+, 100), 192 (20), 174 (81),
164 (15); CIMS, m/ z (intensity) 208 (M+ + 1, 100), 148 (12).
Dioxibrassinin (9): [R]D +21.3 (MeOH; c 0.0024); HPLC tr
) 7.5 min; 1H NMR (300 MHz, CD3OD) δ 7.36 (d, J ) 7.7 Hz,
1H), 7.27 (dd, J ) 7.7, 7.7 Hz, 1H), 7.01 (dd, J ) 7.7, 7.7 Hz,
1H), 6.89 (d, J ) 7.7 Hz, 1H), 4.32 (d, J ) 13.8 Hz, 1H), 3.98
(d, J ) 13.8 Hz, 1H), 2.53 (s, 3H); 13C NMR (125.8 MHz, CD3-
OD) δ 201.9, 180.6, 142.7, 131.1 (2×) 125.9, 123.9, 111.4, 76.3,
18.2; FTIR (cm-1) 3273, 3223, 2918, 2850, 1720, 1714, 1622,
1470, 1378, 1189, 754; EIMS, m/ z (%) 268 (M+, 12), 220 (25),
191 (15), 164 (29), 149 (100); HREIMS, m/ z calcd for
C
11H12N2O2S2 268.0340, found 268.0345.
RESULTS AND DISCUSSION
In initial experiments, we compared the bioactivity
of cyclobrassinin (6) on the brassica pathogens utilized
in this study, R. solani isolate AG 2-1, the nonaggressive
or avirulent (A) P. lingam, and the aggressive or
virulent (V) P. lingam (avirulent and virulent groups
are now considered two different but related species;
Pedras et al., 1995; Taylor et al., 1995), employing a
mycelial growth inhibition assay (Pedras et al., 1997;
Pedras and Khan, 1996). Cyclobrassinin (at a concen-
tration of 5 × 10-4 M) inhibited the mycelial growth of
the blackleg A isolate and the root rot isolate AG 2-1
(∼30% inhibition relative to control cultures after
incubation for 5 days); a slighter inhibitory effect (∼15%
inhibition relative to control cultures after incubation
for 5 days) was observed on the blackleg V isolate at
identical concentration.
In subsequent experiments, the phytoalexin cyclo-
brassinin (6) was incubated with cultures of the root
rot pathogen R. solani to establish a time course for the
biotransformation. Analysis of organic extracts of cul-
ture samples by HPLC indicated a rapid decrease in the
concentration of cyclobrassinin (HPLC retention time,
tr ) 24.5 min) and the sequential appearance of three
constituents with tr ) 18.8, 10.2, and 5.0, min, respec-
tively. Subsequently, to obtain sufficient quantities of
each constituent for both chemical characterization and
bioassay, larger scale fungal cultures incubated with
1
3H), 6.25 (br s, 1H, D2O exchangeable); H NMR (500 MHz,
CD3OD) δ 8.30 (s, 1H), 7.45 (d, J ) 6.9 Hz, 1H), 7.05 (m, 3H);
13C NMR (125.8 MHz, CD2Cl2) δ 179.0, 148.9, 138.0, 128.3,
124.1, 122.2, 115.3, 109.8, 108.4; 13C NMR (125.8 MHz, CD3-
OD) δ 177.6, 153.7, 151.7, 148.7, 140.4, 139.5, 129.9, 126.9,
125.0, 124.9, 124.1, 123.4, 122.5, 121.6, 120.9, 119.6, 115.8,
112.9, 111.7, 110.8, 110.5, 108.6; FTIR (cm-1) 3238, 3128, 3067,
2924, 2852, 1641, 1623, 1549, 1446, 1398, 1303, 1233, 1210,
1014, 739; EIMS, m/ z (%) 176 (M+, 100), 149 (46), 117 (11;
CIMS, m/ z (%) 177 (M+ + 1, 100), 164 (11), 145 (13); HREIMS,
m/ z calcd for C9H8N2S 176.0408, found 176.0412.
Brassilexin (12). Activated carbon (35 mg, 2.9 mmol) was
added to a solution of 3-methylenaminoindole-2-thione (11)
(51.0 mg, 0.29 mmol) in MeOH (2 mL), the reaction mixture
was stirred at room temperature for 24 h and then filtered
through a tight cotton plug, and the solvent was evaporated
under reduced pressure to yield 41 mg of brassilexin (82%
yield): HPLC tr ) 12.1 min; 1H NMR (500 MHz, CD3CN) δ
9.81 (br s, 1H, D2O exchangeable), 8.68 (s, 1H), 7.90 (d, J )
8.0 Hz, 1H), 7.55 (d, J ) 8.0 Hz, 1H), 7.32 (dd, J ) 8.0, 8.0 Hz,
1H), 7.22 (dd, J ) 8.0, 8.0 Hz, 1H); 13C NMR (125.8 MHz, CD3-
OD) δ 160.5, 148.7, 146.0, 128.4, 124.9, 121.5, 121.4, 120.9,
112.9; FTIR (cm-1) 3371, 3274, 3070, 2958, 2924, 2853, 1645,
1619, 1504, 1465, 1368, 1240, 744; EIMS, m/ z (%) 174 (M+,