Reaction of Ferulic Acid with Nitrite
J. Agric. Food Chem., Vol. 46, No. 8, 1998 3315
f
HCOOH 98:2:1 v/v/v) showed a major, fluorescent spot (R 0.4),
two yellow secondary spots (R
spots (R 0.66-0.5, 0.4, 0.2), and a residual brown spot at the
bottom of the plate. HPLC analysis showed a major peak at
20.3 min and numerous minor accompanying peaks. The
residue was chromatographed over 60 g of flash-column silica
gel (Baker, 40 mm) (CH Cl /MeOH/HCOOH, 100:1:0.5 v/v/v)
f
0.7 and 0.17), a number of trace
f
t
R
2
2
and four fractions were collected.
The first fraction (60 mg) afforded the yellow compound at
R
f
0.7 contaminated with traces of a minor impurity (R
The compound was characterized as 2-methoxy-4,6-dinitro-
phenol (9): mp (CH Cl /hexane) 114-116 °C; UV (MeOH) λmax
16, 264, 330 nm; IR (CHCl
f
0.66).
2
2
-1
2
3
) (ν, cm ) 1560, 1345, 1270, 1200;
, 360 MHz) δ 11.19 (1 H, s, OH), 8.69 (1 H, d,
J ) 2.4 Hz), 7.97 (1H, d, J ) 2.4 Hz), 4.08 (3 H, s, CH
O); 13
NMR (CDCl , 90.5 MHz) δ 150.9, 150.6, 139.3, 132.5, 112.6,
11.0, 57.26; EIMS (70 eV) m/z 214 (M , 100), 197 (95), 184
13), 166 (31), 151 (13), 121 (47); HREIMS (70 eV) 214.0222
for C (calcd 214.0225).
The second fraction (100 mg) contained a mixture of at least
four compounds (R 0.66-0.50). After concentration a strong
1
H NMR (CDCl
3
3
C
3
+
1
(
7
6 2 6
H N O
f
characteristic vanilla odor was detected. Vanillin was isolated
by further chromatography as a crude orange solid and
characterized by comparison with a commercial sample of
F igu r e 1. Products formed by the reaction of ferulic acid (1)
with nitrite in acid.
3
-methoxy-4-hydroxybenzaldehyde (vanillin).
1
rings or double bonds. The simple H NMR spectrum
The third fraction (465 mg) afforded a brown residue
contained signals for one methoxyl group (s, 3.99 ppm)
and three uncoupled aromatic proton signals. The
absence of coupling ruled out a position-6 ring substitu-
tion on ferulic acid and suggested that substitution had
containing mostly the fluorescent compound (R
yellow impurities (R 0.36 and 0.50) were also present. A pale
yellow solid was obtained by crystallization of the residue with
CH Cl /MeOH. The filtrate was concentrated and chromato-
f
0.40). Very
f
2
2
1
3
occurred on either position-2 or -3. The C NMR
spectrum contained nine carbon signals, all but the
methoxyl being aromatic. The spectrum also revealed
three methine-bearing carbons, three-oxygen bearing
quaternary carbons, and one carbonyl carbon signal
resonating at 166 ppm. HMBC and HMQC spectral
analyses were used to assign the connectivities of
protons and carbons to one another. Thus, H-3 was
correlated with C-4a and C-4; H-5 with C-4, C-7, and
C-8a; H-8 with C-4a and C-6; and the methoxyl group
with C-6.
graphed again. Thus by repeated chromatography/crystal-
lization, 240 mg of a yellow solid were obtained. The com-
pound was characterized as 7-hydroxy-6-methoxy-1,2(4H)-
benzoxazin-4-one (3): mp (water) 171-175 °C (decomposition);
-1
UV (MeOH) λmax 214, 347 nm; IR (CHCl
3
) (ν, cm ) 1650, 1505,
475, 1445, 1290, 1200, 1140; H NMR (Me CO-d , 360 MHz)
δ 8.18 (1 H, s, H-3), 7.34 (1 H, s, H-5), 6.96 (1 H, s, H-8), 3.99
1
1
2
6
1
3
(
3 2 6
3 H, s, CH O); C NMR (Me CO-d , 90.5 MHz) δ 166.5 (s,
C-4), 159.5 (s, C-8a), 155.9 (s, C-7), 150.9 (d, C-3), 148.4 (s,
C-6), 114.3 (s, C-4a), 103.5 (d, C-5), 101.3 (d, C-8), 56.65 (q,
+
MeO); EIMS (70 eV) m/z 193 (M , 60), 166 (98), 151 (47), 138
(
14), 123 (58), 110 (13), 95 (35), 69 (100), 53 (43); HRFABMS
+
(
3-NBA) m/z (M+H) found 194.0459 (calcd for C
94.0453).
The fourth fraction (290 mg) contained an intractable
9
H
8
NO
4
These connectivities initially caused us to consider
two possible isomeric structures for the major ferulic
acid product as either compound 3 or 5. 3-Methoxy-7-
nitrobicyclo[4,2,0]octan-4-ol (5) was ruled out because
the MS required loss of an HCN equivalent to give a
strong m/z 166 fragment ion. Furthermore, the IR
spectrum contained strong absorption in the range
1
mixture of components that was impossible to resolve.
Meth a n olysis of Com p ou n d 3. A sample of 15 mg of 3
dissolved in 2 mL of MeOH was added to 10 mL of aqueous 1
N NaOH solution. The mixture was stirred at room temper-
ature for 15 min, acidified to pH 2 with 6 N HCl, and extracted
with EtOAc. Concentration of the organic phase yielded 10
mg of an off-white solid. The compound was analyzed without
further purification and characterized as methyl-2,4-dihy-
droxy-5-methoxybenzoate (4): mp 127-129 °C; UV (MeOH)
-
1
1
530-1570 cm , but none for a nitro group. These
results suggested the presence of a conjugated carbonyl
group in the ferulic acid product structure.
Studies of the reactivity of compound 3 toward acid
and base were very informative. The compound was
stable in acid. When compound 3 was stirred at room
temperature in methanolic sodium hydroxide solution,
a single, nonfluorescent compound was rapidly pro-
-
1
λ
max 224, 260, 317 nm; IR (CHCl
1
3
) (ν, cm ) 1670, 1505, 1440,
1
270, 1245, 1200, 1160; H NMR (CDCl , 360 MHz) δ 10.68 (1
H, s, HO-4), 7.22 (1 H, s), 6.53 (1 H, s), 6.13 (1 H, s br, HO-2),
3
1
3
3
9
.92 (3 H, s, CH
0.5 MHz) δ 170.2 (s, CO), 158.5 (s), 152.8 (s), 139.9 (s), 109.9
O-5), 51.97 (q, CH OCO);
EIMS (70 eV) m/z 198 (M , 42), 166 (100), 151 (29), 138 (9),
23 (29), 95 (13), 69 (33); HREIMS (70 eV) m/z 198.0521 for
(calcd 198.0528).
3 3 3
O-5), 3.87 (3 H, s, CH OCO); C NMR (CDCl ,
1
13
duced. H-, C-NMR, and EIMS data suggested that
compound 4 was the methyl ester of 2,4-dihydroxy-5-
methoxybenzoic acid. The EI mass spectra of com-
pounds 3 and 4 were very similar except for their
respective molecular ions. Thus, the structure 7-hy-
droxy-6-methoxy-1,2(4H)-benzoxazin-4-one could be as-
signed as compound 3 for the fluorescent product
obtained by reaction of nitrite with ferulic acid (Figure
1).
(d), 103.3 (s), 103.1 (d), 56.36 (q, CH
3
3
+
1
9 10 5
C H O
RESULTS AND DISCUSSION
Reaction of ferulic acid (1) with nitrite at pH 2 yielded
a complex mixture of compounds. TLC and HPLC
analyses showed the presence of a major, fluorescent
product (3) which was isolated and purified by repeated
chromatography and crystallization and characterized
by spectroscopic analysis.
1,2(4H)-Benzoxazin-4-one derivatives are not com-
mon. However, compatible spectroscopic data were
reported for a dimethoxy substituted derivative (3a )
(S a´ nchez-Viesca and G o´ mez, 1986). Furthermore hy-
drolysis of 5,6,7,8-tetrafluoro-2-phenyl-1,2(4H)benzox-
azin-4-one either in acid or base at high temperature
The HRMS for compound 3 gave C9H8NO4 indicating
a structure with one nitrogen atom and containing seven