Preparative-Scale Synthesis of Two Metabolites
J. Agric. Food Chem., Vol. 50, No. 3, 2002 497
vacuum filtration yielding 59.1 g (61%) of 3-amino-2-hydroxy-3-
1.81-1.74 (m, 2H, H4), 1.21 (s, 3H, CH3), 0.79 (t, 3H, H5). 13C NMR
(75.5 MHz, DMSO-d6): δ 174.6 (C1), 164.2 (CONH), 136.8, 135.6,
134.8, 127.0 (Ph), 72.4 (C2), 59.0 (C3), 27.3 (C4), 19.0 (Ph-CH3), 17.6
(CH3), 8.0 (C5).
methylpentanamide hydrochloride 6. IR (KBr, cm-1): 3415, 3314,
1
3190 (N-H, O-H), 2980, 2921 (C-H), 1672 (CdO). H NMR (300
MHz, D2O): δ 4.19 (s, 1H, H2), 1.88-1.60 (m, 2H, H4), 1.36 (s, 3H,
CH3), 0.99 (t, 3H, H5). 13C NMR (75.5 MHz, D2O): δ 177.8 (C1),
74.0 (C2), 60.6 (C3), 29.1 (C4), 20.3 (CH3), 8.2 (C5).
3-(3,5-Dichloro-benzoylamino)-2-hydroxy-3-methyl-butyric Acid
(14). Compound 14 was prepared in an analogous fashion from 13 as
a white solid in 68% yield; mp ) 162-164 °C (lit mp ) 161-163
3-Amino-2-hydroxy-3-methylbutyramide Hydrochloride (11).
Compound 11 was prepared in analogous fashion from 10 as a white
solid in 32% yield. 1H NMR (200 MHz, D2O): δ 0.3 (s, 3H), 1.35 (s,
3H), 4.1 (s, 1H), 4.7 (s, 7H).
1
°C). H NMR (200 MHz, DMSO-d6): δ 1.4 (d, 6H), 4.5 (s, 1H), 5.6
(s, b, 1H), 7.8 (s, 3H), 8.0 (s, 1H), 12.6 (s, b, 1H).
3-(3,5-Dichloro-4-methyl-benzoylamino)-2-hydroxy-3-methyl-
pentanoic Acid (3). A solution of dimethyl sulfoxide (5.0 g, 0.066
mol) in dichloromethane (46 mL) was added dropwise to a solution of
oxalyl chloride (6.0 g, 0.048 mol) in dichloromethane (73.2 mL) keeping
the temperature below -55 °C. The resulting mixture was stirred for
5 min, and then, a solution of 9 (10.0 g, 0.030 mol) in tetrahydrofuran
(73 mL) was added dropwise over a 20 min period. Stirring was
continued at -50 to -60 °C for 30 min. Triethylamine (15.0 g, 0.150
mol) in dichloromethane (23.2 mL) was added, and the mixture was
stirred for another 10 min. The reaction mixture was allowed to warm
to room temperature over a 30 min period and diluted with water:
dichloromethane (1:1, 1000 mL), and the two layers were separated.
The organic layer was evaporated in vacuo, and water was added to
the residue, acidified to pH 1 with concentrated hydrochloric acid, and
extracted with ethyl acetate. The organic extracts were combined,
dried over anhydrous magnesium sulfate, filtered, evaporated in
vacuo, and washed with hexane to give 5.0 g (50%) of 3 as a white
solid. IR (KBr, cm-1): 3358-3084 (O-H, N-H), 2988, 2974, 2884
(C-H), 1742, 1727 (CdO). 1H NMR (300 MHz, acetone-d6): δ 8.43
(s (D), 1H, NH), 7.85 (s, 2H, Ph), 2.49 (s, 3H, Ph-CH3), 2.20-2.06
(m, 2H, H4), 1.57 (s, 3H, CH3), 0.91 (t, 3H, H5). 13C NMR (75.5 MHz,
acetone-d6): δ 195.8 (C2), 165.4, 163.0 (C1, CONH), 138.4, 135.9,
134.1, 127.6 (aromatic protons), 63.4 (C3), 21.2 (Ph-CH3), 17.6 (CH3),
7.9 (C5).
N-[1-(Carbamoyl-hydroxy-methyl)-1-methyl-propyl]-3,5-dichloro-
4-methyl-benzamide (7). Procedure A. To a solution of sodium
hydroxide (6.6 g, 0.164 mol) and 6 (10.0 g, 0.055 mol) in water (51
mL) was added dropwise a solution of 3,5-dichloro-4-methylben-
zoyl chloride (24.5 g, 0.109 mol) in toluene (66 mL) over a period of
1 h, and the resulting mixture was stirred for 2 h at room tempera-
ture. The solid formed was collected by vacuum filtration, washed
with water, toluene, and methanol, and dried in vacuo at 50 °C over-
night to afford 17.1 g of a yellow solid 1:1 mixture of 3,5-dichloro-
4-methyl-benzoic acid 1-carbamoyl-2-(3,5-dichloro-4-methyl-benzoyl-
amino)-2-methyl-butyl ester (8) and 7. This mixture was suspended in
a solution of sodium hydroxide (1.31 g, 33 mmol) in ethanol (125 mL)
and refluxed for 3 h. The reaction mixture was cooled to room
temperature, and the solvent was removed in vacuo. The residue was
slurred in water, and the solid was collected by vacuum filtration,
washed with toluene, and dried in vacuo at 50 °C overnight to
yield 7.5 g of 7. IR (KBr, cm-1): 3445, 3334, 3283 (N-H, O-H),
1
2974, 2939, 2879 (C-H), 1678, 1650 (CdO). H NMR (200 MHz,
DMSO-d6): δ 8.49 (s (D), 1H, NH), 7.82 (s, 2H, Ph), 7.58 (s, 2H,
NH2), 6.15 (s, 1H, OH), 4.15 (s, 1H, H2), 2.50 (s, 3H, Ph-CH3), 2.14-
1.93 (m, 2H, H4), 1.27 (s, 3H, CH3), 0.81 (t, 3H, H5). 13C NMR (75.5
MHz, DMSO-d6): δ 176.3 (C1), 162.7 (Ph-CO), 136.4, 135.5, 134.5,
126.4 (Ph), 70.9 (C2), 58.9 (C3), 26.4 (C4), 19.2 (Ph-CH3), 17.3 (CH3),
7.7 (C5).
Procedure B. A solution of sodium hydroxide (16.5 g, 0.413 mol)
and 6 (25.0 g, 0.137 mol) in water (128 mL) was added dropwise with
vigorous stirring over a 1 h period to a solution of 3,5-dichloro-4-
methylbenzoyl chloride (15.4 g, 0.069 mol) in toluene (41 mL). After
the addition was completed, the reaction mixture was stirred for 2 h at
room temperature. The resulting solid was collected by vacuum
filtration, washed with water, toluene, and methanol, and dried in vacuo
at 50 °C overnight to give 11.6 g (51%) of a white solid 14:1 mixture
of 7 and an unidentified impurity.
3-(3,5-Dichloro-benzoylamino)-3-methyl-2-oxo-butyric Acid (4).
Compound 4 was prepared in an analogous fashion from 14 as a white
solid in 77% yield; mp ) 155-157 °C. 1H NMR (200 MHz, acetone-
d6): δ 1.6 (s, 6H), 7.7 (m, 1H), 7.85 (m, 2H), 8.5 (s, b, 1H).
LITERATURE CITED
(1) Egan, A. R.; Michelotti, E. L.; Young, D. H.; Wilson, W.;
Mattioda, H. RH-7281: A Novel Fungicide for Control of
Downy Mildew and Late Blight. Brighton Crop Protection
Conference: Pests Diseases; British Crop Protection Council:
Bracknell, U.K., 1998; Vol. 2, pp 335-342.
N-(2-Carbamoyl-2-hydroxy-1,1-dimethyl-ethyl)-3,5-dichloro-ben-
zamide (13). A 2:1 mixture of 3,5-dichloro-benzoic acid 1-carbamoyl-
2-(3,5-dichloro-benzoylamino)-2-methyl-propyl ester (12) and N-(2-
carbamoyl-2-hydroxy-1,1-dimethyl-ethyl)-3,5-dichloro-benzamide was
(2) Compound 3, or 3 -(3,5-dichloro-4-methylbenzamido)-2-oxo-3-
methylpentanoic acid, was recently isolated and identified from
soil metabolism, soil column leaching, and aquatic sediment
studies conducted with 14C-RH-7281. For the soil metabolism
study, metabolically active soil samples were treated with 14C-
RH-7281 to approximate the field rate of 0.2 kg a.i./ha. For the
aged column leaching study, treated soil samples from the soil
metabolism study were loaded onto a column of fresh soil and
leached with water to simulate a rainfall event. For the water
sediment study, water/sediment systems in flasks were incubated
with 14C-RH-7281. For each of these studies, material balance
analyses and identification of metabolites were performed. A
complete report of this information will be reported in later
publications. Compound 3 was isolated as a discrete 14C entity
in each of these studies. Proof of structure by mass spectrometry
required isolation of sufficient quantities of material for HPLC/
MS analyses. Material for mass spectrometer identification was
isolated from the combined samples of three studies. Extracts
from the water phase of the water sediment study, extracts from
soil column segments from the column leaching study, and soil
extracts from the soil metabolism study were combined to obtain
sufficient material. Confirmation of structure was obtained by
chemical synthesis of compound 3 and comparison of mass
spectra and chromatographic behavior.
1
prepared using the same procedure as above in 69% yield. H NMR
(200 MHz, DMSO-d6): mix δ 1.35 (s), 0.4 (s), 1.5 (s), 1.6 (s), 3.4 (s,
b), 4.2 (s), 5.7 (s), 6.1 (s, b), 7.45 (s, b), 7.5 (s, b), 7.7 (s, b), 7.75 (s),
7.95 (t), 8.0 (t), 8.4 (s, b), 8.45 (s, b). Hydrolysis of the ester was carried
1
out in standard procedure to yield quantitatively butyramide 13. H
NMR (200 MHz, DMSO-d6): δ 1.4 (d, 6H), 4.2 (s, b, 1H), 6.0 (s, b,
1H), 7.4 (d, b, 2H), 7.8 (s, 3H), 8.4 (s, b, 1H).
N-[1-(Carbamoyl-hydroxy-methyl)-1-methyl-propyl]-3,5-dichloro-
4-methyl-benzamide (9). To a suspension of 7 (17.0 g, 0.051 mol) in
dioxane (283 mL) was added dropwise concentrated hydrochloric acid
(43 mL, 0.516 mol), followed by portionwise addition of solid sodium
nitrite (35.2 g, 0.510 mol). The resulting mixture was stirred overnight
at room temperature. The reaction mixture was poured into iced water
(680 mL) and extracted with diethyl ether (2 × 340 mL). The combined
organic layers were dried over anhydrous magnesium sulfate, the ether
was removed in vacuo, and water (250 mL) was added to the resulting
residue. The resulting suspension was cooled at 0 °C and 50% sodium
hydroxide solution was added until most of the solid was dissolved
(approximately 180 mL). The suspension was filtered, and the filtrate
was brought to pH 1 with concentrated hydrochloric acid. Trituration
in hexane afforded 12.0 g of 9 (71%) as a light yellow solid. IR (KBr,
cm-1): 3400-3080 (O-H, N-H), 2972, 2939, 2881 (C-H), 1719,
1652 (CdO). 1H NMR (300 MHz, DMSO-d6): δ 8.22 (s (D), 1H, NH),
7.82 (s, 2H, Ph), 4.42 (s, 1H, H2), 2.47 (s, 3H, Ph-CH3), 2.20-2.11,
(3) Yih, R. Y.; Swithenbank, C. J. Agric. Food Chem. 1971, 19,
314-319.