3418 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Wang et al.
) 7.1 Hz, 2H, OCH2CH3), 6.02 (s, 2H, NH2), 6.90 (ddd, J )
1.4 Hz, J ) 8.9 Hz, J ) 8.9 Hz, 1H, aromatic), 7.27-7.34 (m,
1H, aromatic). Found: C, 56.05%; H, 5.07%; N, 5.37%.
Requires: C, 56.03%; H, 5.09%; N, 5.45%.
the precipitate was discarded, and the filtrate was evaporated
under reduced pressure. The resulting white solid residue was
purified using column chromatography by elution with dichlo-
romethane/ethyl acetate 4:1 giving compound 8 (57%) as a
single spot on a TLC plate with Rf ) 0.67 (dichloromethane/
ethyl acetate 4:1). The structure of this active ester was
confirmed by a 1H NMR spectrum to be similar to that of
compound 7 but with the succinimide protons at δ 2.84 (CDCl3)
and mass spectrum m/z 658 (M+1, 9%), 332 (100), 327 (38),
312 (31), 296 (44).
3,5-Dichloro-4-[3-(ethyoxycarbonyl)propyloxy]aniline (9). Into
30 mL of dry dimethyl sulfoxide (DMSO), a mixture of 7.2 g of
4-amino-2,6-dichlorophenol (40.4 mmol), 7.15 g of ethyl 4-bromo-
butyrate (36.6 mmol), and 6.1 g of finely ground of potassium
carbonate (44.1 mmol) was added. The mixture was stirred
under N2 at room temperature for 7 h. At the end of the
reaction, 100 mL of 1 M sodium hydroxide (NaOH) and 150
mL of ether were added. The aqueous layer was washed with
ether, and the combined organic layer was washed with 1 M
NaOH, water, and brine and then dried with Na2SO4. After
removing the solvent under reduced pressure, an oily product
was obtained in 64% yield: TLC Rf ) 0.73 (ethyl acetate/
toluene 1:1); 1H NMR (CDCl3) δ 1.27 (t, J ) 7.1 Hz, 3H,
OCH2CH3), 2.12 (5 line m, J ) 6.5 Hz, 2H, CH2CH2CH2), 2.64
(t, J ) 7.4 Hz, 2H, CH2CH2COO), 3.63 (bs, 2H, NH2), 3.95 (t,
J ) 6.0 Hz, 2H, OCH2CH2CH2), 4.16 (q, J ) 7.1 Hz, 2H, OCH2-
CH3), 6.60 (s, 2H, aromatic). This product was not purified
further before the next reaction.
1-[3,5-Dichloro-4-[3-(ethyoxycarbonyl)propyloxy]phenyl]-3-
(2,6-difluorobenzoyl)urea (10). A solution of 3.1 g of freshly
distilled 2,6-difluorobenzoyl isocyanate (17 mmol) in 10 mL
dry benzene was added to a stirred solution of 5 g of compound
9 (17 mmol) in 20 mL dry benzene under N2. The temperature
was kept below 40 °C in an ice bath. After 6 h, the resulting
crystals were collected, washed with benzene, and dried to
yield 7.7 g of a fine white solid. Recrystallization from
acetonitrile gave compound 10 as white needles (87%): mp
134-136 °C; TLC Rf ) 0.76 (ethyl acetate/toluene 1:1); 1H
NMR (CDCl3) δ 1.28 (t, J ) 7.1 Hz, 3H, OCH2CH3), 2.16 (5
line m, J ) 6.5 Hz, 2H, CH2CH2CH2), 2.66 (t, J ) 7.3 Hz, 2H,
CH2CH2COO), 4.04 (t, J ) 6.0 Hz, 2H, OCH2CH2CH2), 4.17
(q, J ) 7.1 Hz, 2H, OCH2CH3), 7.07 (t, J ) 8.3 Hz, 2H,
aromatic). 7.48 (s, 2H, aromatic), 7.50-7.60 (m, 1H, aromatic),
9.29 (s, 1H, NH), 10.50 (s, 1H, NH).
1-[3,5-Dichloro-4-(3-carboxypropyloxy)phenyl]-3-(2,6-difluo-
robenzoyl)urea (11, Hapten B). A suspension of 3 g of compound
10 (6.3 mmol) and 0.98 g of finely ground potassium hydroxide
in 150 mL of water and 75 mL of methanol was stirred at room
temperature for 3 h. The methanol was evaporated, and the
residue was washed with ethyl acetate (2 × 150 mL). The
aqueous layer was acidified to pH 1 with 1 M HCl, then
extracted with ethyl acetate (2 × 200 mL). The combined
organic layer was washed with water and brine and dried with
Na2SO4, and the solvent was removed to produce 2.1 g of a
white solid. Recrystallized from acetonitrile yielded white
needle crystals of hapten B (67%): mp 173-175 °C; TLC Rf )
0.69 (ethyl acetate); 1H NMR (CDCl3/DMSO) δ 2.14 (5 line m,
J ) 6.5 Hz, 2H, CH2CH2CH2), 2.64 (t, J ) 7.4 Hz, 2H, CH2CH2-
COO), 4.05 (t, J ) 6.0 Hz, 2H, OCH2CH2CH2), 7.02 (t, J ) 8.2
Hz, 2H, aromatic), 7.43-7.54 (m, 1H, aromatic), 7.58 (s, 2H,
aromatic), 10.27 (s, 1H, NH), 10.31 (s, 1H, NH); 13C NMR
(DMSO) δ 25.0, 29.9, 72.6 (CH2CH2CH2), 112.0 (dd, J ) 2.4,
21.8 Hz, C′3 and C′5), 113.3 (t, J ) 21.1 Hz, C′1), 120.6 (s, C2
and C6), 128.2 (s, C3 and C5), 133.2 (t, J ) 10.1 Hz, C′4), 134.5
(s, C1), 146.7 (s, C4), 150.0 (s, NHCONH), 158.6 (dd, J ) 7.0,
250.1 Hz, C′2 and C′6), 161.9 (s, aromatic-CONH), 173.9 (s,
COOH). Found: C, 48.35%; H, 2.96%; N, 6.08%. Requires: C,
48.34%; H, 3.16%; N, 6.26%.
2-Fluoro-4-(2-chloro-4-trifluoromethylphenoxy)aniline (5). A
suspension of 1.6 g of flufenoxuron (3.3 mmol) in 50 mL of
acetic acid and 50 mL of 6 M HCl was stirred and heated in
an oil bath at 120 °C overnight. The solvent was evaporated
under vacuum and the product was redissolved in 200 mL of
ethyl acetate and then extracted with 6 M HCl (3 × 50 mL).
The combined aqueous phase was washed with ethyl acetate
(3 × 50 mL) and 6 M NaOH was added to obtain a pH of about
13. The aniline was then re-extracted with ethyl acetate (3 ×
100 mL). This organic portion was evaporated to yield 1.02 g
of 2-fluoro-4-(2-chloro-4-trifluoromethylphenoxy)aniline as an
oil (54%): TLC Rf ) 0.73 (toluene/methanol 17:3); 1H NMR
(CDCl3/DMSO) δ 3.12 (bs, 2H, NH2), 6.67-6.82 (bd, J ) 8.8
Hz, 1H, aromatic), 7.02 (m, 2H, aromatic), 7.39 (bt, J ) 8.5
Hz, 1H, aromatic), 7.50 (bdd, J ) 2.2 Hz, J ) 8.5 Hz, 1H,
aromatic), 7.74 (d, J ) 2.0 Hz, 1H, aromatic); MS m/z 306
(M+1, 100%), 286 (80), 270 (28), 250 (23), 139 (14).
1-[4-(2-Chloro-R,R,R-trifluoro-4-tolyloxy)-2-fluorophenyl]-3-
[2,6-difluoro-3-(2-ethoxycarbonylethyl)benzoyl]urea (6). Com-
pound 4 was reacted with freshly distilled oxalyl chloride to
make 2,6-difluoro-[2-(ethyoxycarbonyl)ethyl]benzoyl isocyanate
according to the method of Wie et al. (1982). Next, a solution
of the crude isocyanate in dry benzene was added directly to
a stirred solution of compound 5 in dry benzene. The mixture
was stirred overnight at room temperature with the exclusion
of moisture, the solvent was removed by evaporation under
reduced pressure, and the residue was recrystallized from
acetonitrile to give white crystals of compound 6: 1H NMR
(CDCl3) δ 1.23 (t, J ) 7.1 Hz, 3H, OCH2CH3), 2.62 (t, J ) 7.4
Hz, 2H, CH2COO), 2.97 (t, J ) 7.5 Hz, 2H, CH2Ar), 4.11 (q, J
) 7.1 Hz, 2H, OCH2CH3), 6.78 (bd, J ) 8.8 Hz, 1H, aromatic),
6.85 (dd, J ) 2.4, J ) 12.8 Hz, 1H, aromatic), 6.97 (dt, J ) 1.3
Hz, J ) 8.9 Hz, 1H, aromatic), 7.03 (d, J ) 8.7 Hz, 1H,
aromatic), 7.42 (dt, J ) 6.3 Hz, J ) 8.5 Hz, 1H, aromatic),
7.49 (dm, J ) 8.7 Hz, 1H, aromatic), 7.76 (d, J ) 2.2 Hz, 1H,
aromatic), 8.05 (t, J ) 8.8 Hz, 1H, aromatic), 9.16 (s, 1H, NH),
10.69 (s, 1H, NH).
1-[4-(2-Chloro-R,R,R-trifluoro-4-tolyloxy)-2-fluorophenyl]-3-
[2,6-difluoro-3-(2-carboxyethyl)benzoyl]urea (7, Hapten A). The
hydrolysis reaction of compound 6 (0.6 g) was conducted in
the aqueous methanol (1:1) with 2 equiv of KOH. After 7 h,
the reaction mixture was extracted with chloroform (3 × 60
mL). The aqueous phase was acidified with 6 M HCl to pH 2
and extracted with chloroform (3 × 60 mL). The combined
organic layers were then dried with Na2SO4 and concentrated
under reduced pressure. The residue was purified using
column chromatography on silica gel (ethyl acetate, 10%
methanol in ethyl acetate) to give hapten A as a pale-yellow
solid (0.254 g): 1H NMR (CDCl3) δ 2.71 (t, J ) 7.4 Hz, 2H,
CH2COO), 2.98 (t, J ) 7.3 Hz, 2H, CH2Ar), 6.82 (bd, J ) 8.8
Hz, 1H, aromatic), 6.87 (dd, J ) 2.6, J ) 11.3 Hz, 1H,
aromatic), 6.97 (dt, J ) 1.1 Hz, J ) 8.9 Hz, 1H, aromatic),
7.05 (d, J ) 8.5 Hz, 1H, aromatic), 7.39 (dt, J ) 6.3 Hz, J )
8.5 Hz, 1H, aromatic), 7.50 (dm, 1H, aromatic), 7.76 (d, J )
2.1 Hz, 1H, aromatic), 8.16 (t, J ) 8.7 Hz, 1H, aromatic), 10.34
(s, 1H, NH), 10.81 (s, 1H, NH); 13C NMR (CDCl3) δ 23.3, 33.3
(s, CH2CH2), 106.3 (d, C3), 110.9 (dd, C′5), 112.5 (t, C′1), 114.3
(d, C5), 118.8 (s, C6), 120.9 (s, C′′2), 122.2 (s, C′′6), 122.4 (d,
C1), 123.7 (dd, C′3), 124.8 (q, C′′4 and C′′5), 125.3 (q, CF3), 127.5
(q, C′′3), 132.7 (t, C′4), 150.0 (s, CONH-Ar), 150.9 (s, C4), 154.6
(d, C2), 154.9 (s, C′′1), 155.4 (q, C′2), 158.7 (q, C′6), 162.6 (s,
Ar-CONH), 173.2 (s, COOH); MS m/z 561 (M+1, 13%), 332
(100), 306 (29), 230 (91), 213 (45).
Preparation of the Active Ester of 1-[4-(2-Chloro-R,R,R-
trifluoro-4-tolyloxy)-2-fluorophenyl]-3-[2,6-difluoro-3-(2-carboxy-
ethyl)benzoyl]urea (8). The N-hydroxysuccinimide (NHS) ester
of hapten A was formed as follows. The hapten (0.36 g) was
combined with NHS (0.081 g) in freshly distilled tetrahydro-
furan (THF, 15 mL), and then N,N-dicyclohexylcarbodiimide
(DCC, 0.145 g) was added with stirring. The reaction was
allowed to run overnight under N2. The mixture was filtered,
Preparation of Active Ester of 1-[3,5-Dichloro-4-(3-carboxy-
propyloxy)phenyl]-3-(2,6-difluorobenzoyl)urea (12). The active
ester of compound 11 was prepared as for compound 8.
Purification using column chromatography by elution with
dichloromethane/ethyl acetate 4:1 gave compound 12 (74%):
1
TLC Rf ) 0.45 (dichloromethane/ethyl acetate 4:1); H NMR
(CDCl3/DMSO) δ 2.14 (5 line m, J ) 6.5 Hz, 2H, CH2CH2CH2),