New bitter-masking compounds: Hydroxylated benzoic acid amides of aromatic amines as structural analogues of homoeriodictyol

Article abstract of DOI:10.1021/jf0617061

Starting from the known bitter-masking flavanones eriodictyol and homoeriodictyol from herba santa some structurally related hydroxybenzoic acid amides of benzylamines were synthesized and evaluated as masking agents toward bitterness of caffeine by senso

Full text of DOI:10.1021/jf0617061

8574 J. Agric. Food Chem. 2006, 54, 8574−8579
New Bitter-Masking Compounds: Hydroxylated Benzoic Acid
Amides of Aromatic Amines as Structural Analogues of
Homoeriodictyol
JAKOB P. LEY,* MARIA BLINGS, SUSANNE PAETZ, GERHARD E. KRAMMER, AND
HEINZ-J U¨ RGEN BERTRAM
Symrise GmbH & Co. KG, Flavor & Nutrititon Research & Innovation, P.O. Box 1253, 37601
Holzminden, Germany
Starting from the known bitter-masking flavanones eriodictyol and homoeriodictyol from herba santa
some structurally related hydroxybenzoic acid amides of benzylamines were synthesized and
evaluated as masking agents toward bitterness of caffeine by sensory methods. The closest structural
relatives of homoeriodictyol, the hydroxybenzoic acid vanillylamides 5-9, were the most active and
-
1
were able to reduce the bitterness of a 500 mg L caffeine solution by about 30% at a concentration
of 100 mg L-1. 2,4-Dihydroxybenzoic acid vanillylamide 7 showed a clear dose-dependent activity
as inhibitor of the bitter taste of caffein between 5 and 500 mg L-1. Additionally, it was possible to
reduce the bitterness of quinine and salicine but not of the bitter peptide N-L-leucyl-L-tryptophan.
Combinations of homoeriodictyol and amide 7 showed no synergistic or antagonistic changes in
activity. The results for model compound 7 suggested that the hitherto unknown masking mechanism
is probably the same for flavanones and the new amides. In the future, the new amides may be
alternatives for the expensive flavanones to create flavor solutions to mask bitterness of pharma-
ceuticals or foodstuffs.
KEYWORDS: Bitter masking; taste modifiers; hydroxylated benzoic acid amides; homoeriodictyol;
structure-activity relationship
INTRODUCTION
were discussed in this study. To get more information about
the mechanism of inhibition, we have started a synthesis
program to yield structural analogues of flavanones. Further-
more, the availability from nature of the most active flavanones
eriodictyol and especially homoeriodictyol is limited. Some
syntheses of these flavanones are known (11, 12) but they mostly
need multistep procedures using expensive or toxic raw materi-
als. Therefore, as a first step we decided to investigate
hydroxylated benzoic acid amides of benzylamines as structural
analogues, which are much simpler to synthesize but contain
the most important structural elements of the original flavanones
(cf. Figure 1).
Pronounced bitter taste is mostly not tolerated in food and
wanted only in exceptional cases, e.g., in coffee, dark chocolate,
or tea. Frequently, the compounds that are responsible for the
bitter, astringent, or harsh taste are either eliminated from raw
materials and foodstuffs or masked by different techniques. In
contrast, a lot of bitter-tasting phytonutrients, such as poly-
phenols, flavonoids, isoflavones, terpenes, and glucosinolates,
are supposed to be healthy (1). Significant progress regarding
the understanding of bitter taste reception on the molecular level
was made during the past decade (2) and nowadays the agonist
activity of several bitter molecules on bitter receptors is known
(
3-6).
Some hydroxylated benzoic acid amides of benzylamines
were known [e.g., 3,4-dihydroxybenzoic acid N-(3,4-dihydroxy-
benzyl)amide as an amyloid protection agent (13)], but amides
showing a substitution pattern closest to the most active
flavanones were not described yet. In the investigated structural
class only a few naturally occurring amides of phenethylamines
are known from the literature: aduncamide [3, 2-hydroxy-5-
methoxybenzoic acid N-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-
amide, cf. Figure 2] from Piper aduncum (14) and some salicoyl
tyramines such as 2-hydroxybenzoic acid N-[2-(4-hydroxyphen-
yl)ethyl]amide (4) from Naiba riparia (15, 16). The closest
known relative is 2,4,6-trihydroxybenzoic acid N-(3-hydroxy-
Recently, some progress was made in the identification of
molecules that are able to cover or mask some of the bad tastes
caused by several bitter substances, e.g., Maillard reaction
products related to alapyraidine (7), γ-aminobutyric acid (8),
and nucleotides such as AMP (9). In our previous study we
have shown that some flavanones, especially homoeriodicyol
(1) and eriodictyol (2), are the main bitter-masking actives from
herba santa (Eriodictyon californicum) (10); in addition, some
of the first structure-activity relationships for several flavanones
*
To whom correpondence should be addressed. Phone: Int. + 49 5531
9
0 1883. Fax: Int. + 49 5531 90 48883. jakob.ley@symrise.com.
1
0.1021/jf0617061 CCC: $33.50
© 2006 American Chemical Society
Published on Web 10/06/2006

Hydroxylated Benzoic Acid Amides as Bitter-Masking Compounds
J. Agric. Food Chem., Vol. 54, No. 22, 2006 8575
Germany). Flash chromatography (FC) was performed on a Biotage
Flash 40 system (Biotage AB, Uppsala, Sweden) using disposable prep-
packed columns.
Syntheses of Hydroxylated Benzoic Acid Amides, General
Procedure. The hydroxylated benzoic acid (10-20 mmol), N-hydroxy-
succinimide (1 mol equiv), and N,N′-dicyclohexylcarbodiimide (1 mol
equiv) were dissolved in 1,4-dioxane (2.0 mL/mmol) while stirring
under nitrogen atmosphere. The turbid mixture was stirred at 20-25 °C
for 16 h. The precipitate was filtered off and the filtrate directly added
to a solution of the amine or its hydrochloride (1.1 mol equiv) and
Figure 1. Structural relationship between homoeriodictyol (1), eriodictyol
(2), and hydroxylated benzamides of benzylamines.
3
NaHCO (1 mol equiv) in water (1 mL/mmol). The reaction mixture
was heated to 50 °C under nitrogen and stirred for 1 h. After cooling
to room temperature, dilute HCl (10%) was added and the mixture
was extracted using ethyl acetate. The combined organic phases were
2 4
washed with brine, dried with anhydrous Na SO , and filtered, and the
filtrate was evaporated in vacuo. In some cases the aryl esters were
found as minor byproducts. In such cases it was of advantage to
saponify the crude product using 25% NaOH at 40 °C and subsequent
treatment with 10% HCl and workup as described earlier. Final
purification was done by FC on silica gel 60, using ethyl acetate/n-
hexane mixtures and/or recrystallization when appropriate.
2
-Hydroxy-5-methoxybenzoic Acid N-2-(4-Hydroxy-3-methoxy-
phenyl)ethylamide, Aduncamide (3). Starting from 10 mmol of
-hydroxy-5-methoxybenzoic acid and 2-(4-hydroxy-3-methoxyphenyl)-
2
ethylamine hydrochloride (11 mmol), the crude product (2.7 g) was
purified by FC (eluant n-hexane/ethyl acetate 3:1 (v/v)) to yield 0.58
g (18%) of colorless crystals. HPLC-MS (RP-18, H
2
O/acetonitrile 80:
16.0 min, m/z )
18.14 (100%, [M + H] ), >95%. H NMR (400 MHz, CDCl ): δ
1.76 (1H, s, OH), 7.00 (1H, dd, J ) 9.0 Hz, J ) 2.9 Hz, H-4), 6.92
2
3
1
0 to 0:100 in 30 min, 15 min isocratic, APCI+): t
R
+
1
3
(
1H, dd, J ) 9.0 Hz, J ) 0.4 Hz, H-3), 6.87 (1H, d, J ) 8.5 Hz, H-5′),
.74-6.70 (2H, m), 6.69-6.67 (1H, m), 6.33 (1H, bs, NH), 5.60 (1H,
s, OH), 3.85 (3H, s, OCH ), 3.73 (3 H, s, OCH ), 3.66 (2H, dt, J ) 7.0
Hz, J ) 6.8 Hz, H-8′), 2.86 (2H, t, J ) 6.87 Hz, H-7′) ppm. C NMR
6
3
3
1
3
(
(
1
100 MHz; CDCl
C), 144.50 (C), 130.27 (C, C-1′), 121,39 (CH, C-6′), 120.89 (CH, C-4),
19.30 (CH, C-3), 114.58 (CH, C-5′), 114.19 (C, C-1), 111.22 (CH,
C-2′), 109.62 (CH, C-6), 55.99 (CH , OCH ), 55.91 (CH , OCH ), 40.92
CH , C-8′), 35.14 (CH , C-7′) ppm.
-Hydroxybenzoic Acid N-2-(4-Hydroxyphenyl)ethylamide (4).
3
): δ 169.63 (C, C-7), 155.51 (C), 151.76 (C), 146.79
3
3
3
3
(
2
2
2
Starting from 10 mmol of 2-hydroxybenzoic acid and 2-(4-hydroxy-
phenyl)ethylamine (11 mmol), the crude product was purified by FC
(
eluant n-hexane/ethyl acetate 1:1 (v/v)) to yield 0.91 g (35%) of
colorless crystals. HPLC-MS (RP-18, H O/acetonitrile 80:20 to 0:100
in 30 min, 15 min isocratic, APCI+): t 14.5 min, m/z ) 258.18 (100%,
M + H] ), >95%. H NMR (400 MHz, DMSO-d ): δ 9.18 (1H, s,
2
R
+
1
[
6
Figure 2. Structures of investigated hydroxylated benzamides.
OH), 8.85 (1H, bt, J ) 5.7 Hz, NH), 7.82 (1H, dd, J ) 7.9 Hz, J ) 1.7
Hz, H-6), 7.39 (1H, ddd, J ) 8.2 Hz, J ) 7.3 Hz, J ) 1.7 Hz, H-4),
4-methoxybenzyl)amide, which was synthesized in a study
7
.04 (2H, m, H-2′ and H-6′), 6.89 (1H, dm, J ) 7.6. Hz, H-3 or H-5),
covering structural analogues of the intensely sweet neohes-
peredin dihydrochalcone, but the compound that is substituted
analogously to hesperetin showed no sweetness at all (17).
6.88 (1H, dd, J ) 7.2 Hz, J ) 1.2 Hz, H-5 or H3) 6.68 (2H, m, H-3′
and H-5′), 3.46 (3H, ddm, J ) 7.7 Hz, J ) 5.9 Hz, H-8′), 2.74 (2H, t,
J ) 7.5 Hz, H-7′) ppm. 13C NMR (100 MHz; DMSO-d
): δ 168.69
6
(
C, C-7), 159.93 (C-2), 155.61 (C-4′), 133.49 (CH, C-4), 129.42 (2 ×
CH, C-2′ and C-6′), 129.14 (C, C-1′), 127.56 (CH, C-6), 118.44 (CH,
C-3 or C-5), 117.26 (CH, C-5 or C-3), 115.16 (C, C-1), 115.07 (2 ×
MATERIALS AND METHODS
Dipeptide N-L-leucyl-L-tryptophan (H-Leu-Trp-OH) was obtained
from Bachem (Weil am Rhein, Germany). 4-Hydroxybenzylamine
hydrochloride and 3-hydroxy-4-methoxybenzylamine hydrochloride
were synthesized analogously to the procedure described earlier starting
from the corresponding aldehydes (18). All other chemicals were from
Sigma-Aldrich (Deisenhofen, Germany), Acros (Schwerte, Germany),
or Lancaster Synthesis (Frankfurt, Germany). NMR spectra were
2 2
CH, C-3′ and C-5′), 40.80 (CH , C-8′), 33.95 (CH , C-7′) ppm.
2-Hydroxybenzoic Acid N-(4-Hydroxy-3-methoxybenzyl)amide
(5). Starting from 10 mmol of 2-hydroxybenzoic acid and 4-hydroxy-
3-methoxybenzylamine hydrochloride (11 mmol), the crude product
was purified by FC (eluant n-hexane/ethyl acetate 1:1 to 1:2 (v/v)) to
yield 1.87 g (68%) of colorless crystals. HPLC-MS (RP-18, H
2
O/
acetonitrile 100:0 to 0:100 in 60 min, APCI+): t
R
15.1 min, m/z )
1
+
recorded using a Varian VXR400S ( H, 400 MHz) spectrometer
273.90 (100%, [M + H] ), >95%. HRMS: calcd for C15
H15NO
4
1
(Varian, Darmstadt, Germany) at 25 °C using tetramethylsilane as
6
273.1001, found 273.0979. H NMR (400 MHz, DMSO-d ): δ 9.26
internal standard. LC-MS spectra were recorded using the LCQ HPLC
system Finnigan MAT HP1100 (Finnigan MAT, Egelsbach, Germany;
APCI, atmospheric pressure chemical ionization). Elemental analysis
(1H, t, J ≈ 6 Hz, NH), 8.89 (1H, bs, OH), 7.88 (1H, ddd, J ) 7.9 Hz,
J ) 1.7 Hz, J ) 0.4 Hz, H-6), 7.39 (1H, ddd, J ) 8.3 Hz, J ) 7.2 Hz,
J ) 1.7 Hz, H-4), 6.92 (1H, bs, H-2′), 6.89 (1H, dd, J ) 8.3 Hz, J )
1.2 Hz, H-3), 6.88 (1H, ddd, J ) 8.0 Hz, J ) 7.3 Hz, J ) 1.2 Hz,
H-5), 6.73 (2H, m, H-5′, H-6′), 4.40 (2H, bd, J ) 5.7 Hz, H-7′), 3.84
(combustion analysis for C, H, O and AAS for sodium) was performed
by Bayer Technology Services, Germany. Sodium content was deter-
mined by capillary electrophoresis using inverse detection with imidazol
on a Agilent 3DCE G1600AX equipment (Agilent, Waldbronn,
(3H, s, OCH
C-7), 160.00 (C), 147.37 (C), 145.52 (C), 133.59 (C, C-4), 129.58 (C),
) ppm. 13C NMR (100 MHz; DMSO-d
): δ 168.63 (C,
3 6

8576 J. Agric. Food Chem., Vol. 54, No. 22, 2006
Ley et al.
1
27.68 (CH, C-6), 119.83 (CH), 118.48 (CH), 117.30 (CH), 115.19
2,4-Dihydroxybenzoic Acid N-(3,4-Dihydroxybenzyl)amide (10).
Starting from 20 mmol of 2,4-dihydroxybenzoic acid and 3,4-
dihydroxybenzylamine hydrobromide (22 mmol), the crude product was
purified by FC (eluant n-hexane/ethyl acetate 3:1 (v/v)) to yield 1.3 g
(
(
CH, C-5′), 115.19 (C), 111.87 (CH, C-2′), 55.49 (CH
CH , C-7′) ppm.
-Hydroxybenzoic Acid N-(4-Hydroxy-3-methoxybenzyl)amide
6). Starting from 10 mmol of 4-hydroxybenzoic acid and 4-hydroxy-
-methoxybenzylamine hydrochloride (11 mmol), the crude product
was purified by FC (eluant n-hexane/ethyl acetate 1:1 (v/v)) to yield
3 3
, OCH ), 42.13
2
4
(
3
2
(29%) colorless crystals. HPLC-MS (RP-18, H O/acetonitrile 80:20
to 0:100 in 30 min and then 15 min isocratic, APCI+): t
R
10.34 min,
+
m/z ) 276.0 (100%, [M + H] ). HRMS: calcd for C14
H
13NO
5
1
1
1
.0 g (37%) of colorless crystals. HPLC-MS (RP-18, H
2
O/acetonitrile
6
275.0794, found 275.0785. H NMR (400 MHz, DMSO-d ): δ 10.05
00:0 to 0:100 in 60 min, APCI+): t 15.1 min, m/z ) 273.92 (66%,
R
(1H, s, OH), 8.93 (1H, t, J ) 6.0 Hz, NH), 8.85 (1H, s, OH), 8.72 (1H,
s, OH), 7.72 (1H, d, J ) 8.8 Hz, H-6), 6.71 (1H, d, J ) 2.1 Hz, H-2′),
6.66 (1H, d, J ) 8.1 Hz, H-5′), 6.56 (1H, dd, J ) 8.1 Hz, J ) 2.1 Hz,
H-6′), 6.28 (1H, dd, J ) 8.7 Hz, J ) 2.4 Hz, H-5), 6.23 (1H, d, J )
2.4 Hz, H-3), 4.29 (2H, d, J ) 5.9 Hz, H-7′) ppm. 13C NMR (100
MHz; DMSO-d ): δ 169.04 (C, C-7), 162.52 (C, C-4), 162.13 (C-2),
6
145.00 (C, C-3′), 144.12 (C, C-4′), 129.90 (C, C-1′), 128.83 (CH, C-6),
118.15 (CH, C-6′), 115.23 (CH, C-5′), 114.73 (CH, C-2′), 106.89 (CH,
+
+
[
M + H] ), 546.54 ([2M + H] ) >95%. HRMS: calcd for C15H -
15
1
4 6
NO 273.1001, found 273.273.0996. H NMR (400 MHz, DMSO-d ):
δ 8.63 (1H, bd, J ) 5.8 Hz, NH), 7.74 (2H, dm, J ) 8.5 Hz, H-2,
H-6), 6.88 (1H, m, H-2′), 6.78 (2H, dm, J ) 8.8 Hz, H-3, H-5), 6.69
(
2H, m, H-5′, H-6′), 4.32 (2H, bd, J ) 5.9 Hz, H-7′), 3.73 (3H, s,
1
3
OCH
3
) ppm. C NMR (100 MHz; DMSO-d
6
): δ 165.67 (C, C-7),
1
59.99 (C), 147.27 (C), 145.25 (C), 130.72 (C), 129.04 (2×CH, C-2,
C-6), 125.11 (C), 119.67 (CH, C-6′), 115.07 (CH, C-5′), 114.67 (2×CH,
2
C-5), 102.61 (CH, C-3), 41.62 (CH , C-7′) ppm.
C-3, C-5), 111.75 (CH, C-2′), 55.49 (CH
3
, OCH
3
2
), 42.13 (CH , C-7′)
2,4-Dihydroxybenzoic Acid N-(4-Hydroxybenzyl)amide (11).
Starting from 20 mmol of 2,4-dihydroxybenzoic acid and 4-hydroxy-
benzylamine hydrochloride (22 mmol), the crude product was purified
as described for compound 7 to yield 1.39 g (54%) of colorless crystals.
ppm.
2
,4-Dihydroxybenzoic Acid N-(4-Hydroxy-3-methoxybenzyl)-
amide (7). Starting from 20 mmol of 2,4-dihydroxybenzoic acid and
-hydroxy-3-methoxybenzylamine hydrochloride (22 mmol), the crude
4
2
HPLC-MS (RP-18, H O/acetonitrile 90:10 to 0:100 in 30 min and
product was saponified as described earlier; after addition of dilute
HCl the product precipitated and was filtered to yield 1.49 g (64%) of
colorless crystals. HPLC-MS (RP-18, H
in 30 min and then 15 min isocratic, APCI+): t
2
2
standard TMS): δ 7.62 (1H, d, J ) 8.7 Hz, H-6), 6.93 (1H, d, J ) 1.8
Hz, H-2′), 6.78 (1H, dd, J ) 8.1 Hz, J ) 1.9 Hz, H-6′), 6.75 (1H, d,
J ) 8.1 Hz, H-5′), 6.32 (1H, dd, J ) 8.7 Hz, J ) 2.4 Hz, H-5), 6.29
then 15 min isocratic, APCI+): t
R
15.05 min, m/z ) 259.89 (100%,
259.0845, found 259.0827.
): δ 10.05 (1H, s, OH), 9.29 (1H, s,
+
[M + H] ). HRMS: calcd for C14
H
13NO
4
1
O/acetonitrile 95:5 to 0:100
H NMR (400 MHz, DMSO-d
6
2
16.6 min, m/z )
OH), 8.96 (1H, bt, J ) 6.0 Hz, NH), 7.71 (1H, d, J ) 8.8 Hz, H-6),
7.12 (2H, m, H-2′ and H-6′), 6.71 (2H, m, H-3′ and H-5′), 6.28 (1H,
ddd, J ) 8.7 Hz, J ) 2.4 Hz, J ) 0.8 Hz, H-5), 6.23 (1H, dd, J ) 2.4
R
+
89.87 (100%, [M + H] ), >95%. HRMS: calcd for C15
H15NO
5
1
3
89.0950, found 289.0927. H NMR (400 MHz, CD OD, internal
1
3
Hz, J ) 0.84 Hz, H-3), 4.35 (1H, d, J ) 5.8 Hz, H-7′) ppm. C NMR
(100 MHz; DMSO-d ): δ 169.07 (C, C-7), 162.50 (C, C-4 or C-2),
6
162.15 (C, C-2 or C-4), 156.22 (C, C-4′), 129.28 (C, C-1′), 128.86
(CH, C-6), 128.57 (2×CH, C-2′ and C-6′), 114.95 (2×CH, C-3′ and
C-5′), 106.91 (CH, C-5), 106.49 (C, C-1), 102.62 (CH, C-3), 41.58
(
3
1H, d, J ) 2.4 Hz, H-3), 4.45 (2H, bs, H-7′), 3.83 (3H, s, OCH )
1
3
ppm. C NMR (100 MHz; CD OD): δ 171.06 (C, C-7), 163.82 (C,
3
C-2), 163.52 (C, C-4), 149.05 (C, C-3′), 146.82 (C, C-4′), 131.70 (C,
2
(CH , C-7′) ppm.
C-1′), 130.27 (CH, C-6), 121.38 (CH, C-6′), 116.16 (CH, C-5′), 112.47
2-Hydroxy-4-methoxybenzoic Acid N-(4-Hydroxy-3-methoxy-
benzyl)amide (12). Starting from 13.3 mmol of 2-hydroxy-4-meth-
oxybenzoic acid and 4-hydroxy-3-methoxybenzylamine hydrochloride
(14.6 mmol), the crude product was purified by recrystallization from
ethyl acetate to yield 1.45 g (33%) of colorless crystals. HPLC-MS
(
(
CH, C-2′), 108.82 (C, C-1), 108.48 (CH, C-5), 103.94 (CH, C-3), 56.38
CH , OCH ), 43.80 (CH , C-7′) ppm.
,4-Dihydroxybenzoic Acid N-(4-Hydroxy-3-methoxybenzyl)-
3
3
2
2
amide, Sodium Salt (8). 2,4-Dihydroxybenzoic acid N-(4-hydroxy-3-
methoxybenzyl)amide (7) (260 mg, 0.9 mmol) was dissolved in NaOH
(RP-18, H
2
O/acetonitrile 80:20 to 0:100 in 30 min and then 15 min
-
1
+
(
1 mol L , 0.9 mL), water (1 mL), and ethanol (2 mL) and stirred at
isocratic, APCI+): t
R
15.91 min, m/z ) 304.11 (100%, [M + H] ).
1
50 °C for 1 h. The reaction mixture was evaporated to dryness in vacuo
HRMS: calcd for C16
H
17NO
5
303.1107, found 303.1078. H NMR (400
at 40 °C, the residue triturated with ethyl acetate (10 mL), and the
product filtered and dried to yield 0.246 g of pale yellow crystals.
Sodium content (CE, inverse detection at 350/380 nm, polarity positive,
6
MHz, DMSO-d ): δ 9.07 (1H, t, J ) 5.93 Hz, NH), 8.87 (1H, s, OH),
7.83 (1H, d, J ) 8.9 Hz, H-6), 6.91 (1H, s, H-2′), 6.725 (1H, s, H-5′
or H-6′), 6.720 (1H, s, H-6′ or H-5′), 6.47 (1H, dd, J ) 8.9 Hz, J )
U ) 28 kV, fused silica 50 µm × 5.6 cm): calcd for C15
H
14NNaO
5
2.5 Hz, H-5), 6.42 (1H, d, J ) 2.5 Hz, H-3), 4.38 (1H, d, J ) 5.9 Hz,
1
13
7
.4%, found 7.65%. H NMR (400 MHz, CD
3
OD): δ 7.62 (1H, d, J
H-7′), 3.79 (3H, s, OCH
MHz; DMSO-d
3
), 3.75 (3H, s, OCH
3
) ppm. C NMR (100
)
8.7 Hz, H-6), 6.95 (1H, d, J ) 1.8 Hz, H-2′), 6.79 (1H, dd, J ) 8.1
6
): δ 168.93 (C, C-7), 163.47 (C, C-4), 162.55 (C, C-2),
Hz, J ) 1.9 Hz, H-6′), 6.73 (1H, d, J ) 8.1 Hz, H-5′), 6.10 (1H, dd,
J ) 8.7 Hz, J ) 2.4 Hz, H-5), 6.03 (1H, d, J ) 2.4 Hz, H-3), 4.48
147.34 (C, C-3′), 145.48 (C, C-4′), 129.71 (C, C-1′), 128.71 (CH, C-6),
119.80 (CH, C-6′), 115.17 (CH, C-5′), 111.85 (CH, C-2′), 107.65 (C,
1
3
(
2H, bs, H-7′), 3.83 (3H, s, OCH
3
) ppm. C NMR (100 MHz, CD
3
-
C-1), 105.95 (CH, C-5), 101.06 (CH, C-3), 55.48 (CH
(CH , OCH ), 41.96 (CH , C-7′) ppm.
3 3
, OCH ), 55.25
OD): δ 171.88 (C, C-7), 170.39 (C, C-2), 166.03 (C, C-4), 149.37 (C,
C-3′), 147.45 (C, C-4′), 131.71 (C, C-1′), 131.35 (CH, C-6), 121.30
3
3
2
2.4-Dihydroxybenzoic Acid N-2-(4-Hydroxy-3-methoxyphenyl)-
ethylamide (13). Starting from 20 mmol of 2,4-dihydroxybenzoic acid
and 2-(4-hydroxy-3-methoxyphenyl)ethylamine hydrochloride (22 mmol),
the crude product was purified by FC (eluant n-hexane/ethyl acetate
3:1 (v/v)) to yield 2.1 g (41%) of colorless crystals. HPLC-MS (RP-
(
CH, C-6′), 116.34 (CH, C-5′), 112.33 (CH, C-2′), 110.62 (C, C-1),
07.51 (CH, C-5), 106.16 (CH, C-3), 56.37 (CH , OCH ), 43.82 (CH
C-7′) ppm.
,4,6-Trihydroxybenzoic Acid N-(4-Hydroxy-3-methoxybenzyl)-
amide (9). Starting from 20 mmol of 2,4,6-trihydroxybenzoic acid and
-hydroxy-3-methoxybenzylamine hydrochloride (22 mmol), the crude
product was purified as described for compound 7 to yield 2.74 g (45%)
O/MeOH 50:50 to 5:95
1
3
3
2
,
2
2
18, H O/acetonitrile 90:10 to 0:100 in 30 min and then 15 min isocratic,
4
APCI+): t
R
16.34 min, m/z ) 303.95 (100%, [M + H]+), 606.26
+
(0.4%, [2M + H] ) >95%. HRMS: calcd C16
H17NO
5
303.1107, found
1
of colorless crystals. HPLC-MS (RP-18, H
2
303.1111. H NMR (400 MHz, CD
3
OD): δ 7.54 (1H, dd, J ) 8.7 Hz,
in 15 min and then 15 min isocratic, APCI+): t
R
17.07 min, m/z )
J ) 0.4 Hz, H-6), 6.81 (1H, dt, J ) 1.9 Hz, J ) 0.3 Hz, H-2′), 6.72
(1H, dd, J ) 8.0 Hz, J ) 0.3 Hz, H-5′), 6.67 (1H, ddt, J ) 8.0 Hz, J
) 1.9 Hz, J ) 0.5 Hz, H-6′), 6.30 (1H, dd, J ) 8.7 Hz, J ) 2.4 Hz,
+
3
3
05.91 (100%, [M + H] ), >95%. HRMS: calcd for C15
H
15NO
6
1
05.0899, found 305.0903. H NMR (400 MHz, CD OD): δ 6.93 (1H,
3
d, J ) 1.6 Hz, H-2′), 6.79 (1H, dd, J ) 8.0 Hz, J ) 1.7 Hz, H6′), 6.76
H-5), 6.27 (1H, dd, J ) 2.4 Hz, J ) 0.4 Hz, H-3), 3.78 (3H, d, J ) 0.3
), 3.53 (2H, m, H-8), 2.80 (2H, m, H-7) ppm. 1 C NMR (100
3
3
(
1H, dd, J ) 8.0 Hz, J ) 0.5 Hz, H-5′), 5.85 (2H, s, H-3, H-5), 4.45
Hz, OCH
MHz; CD
3
1
3
(2H, bs, H-7′), 3.84 (3H, s, OCH
3
) ppm. C NMR (100 MHz; CD
3
-
OD): δ 171.15 (C, C-7), 163.79 (C, C-2), 163.41 (C, C-4),
OD): δ 171.85 (C, C-7), 163.48 (C, C-3, C-6), 163.20 (C, C-4), 149.14
148.95 (C, C-3′), 146.05 (C, C-4′), 130.25 (CH, C-6), 122.33 (CH,
C-6′), 116.24 (CH, C-5′), 113.52 (CH, C-2′), 108.91 (C, C-1), 103.92
(C, C-3′), 146.92 (C, C-4′), 131.31 (C, C-1′), 121.31 (CH, C-6′), 116.28
(
5
CH, C-5′), 112.35 (CH, C-2′), 96.99 (C, C-1), 96.02 (CH, C-3, C-5),
6.38 (CH , OCH ), 43.67 (CH , C-7′) ppm.
3 3 2
(CH, C-3), 56.31 (CH
ppm.
3 3 2 2
, OCH ), 42.37 (CH , C-8), 36.30 (CH , C-7)

Hydroxylated Benzoic Acid Amides as Bitter-Masking Compounds
J. Agric. Food Chem., Vol. 54, No. 22, 2006 8577
2
,4-Dihydroxybenzoic Acid N-(3-Hydroxy-4-methoxy)benzyl-
amide (14). Starting from 20 mmol of 2,4-dihydroxybenzoic acid and
-hydroxy-4-methoxybenzylamine hydrochloride (22 mmol), the crude
product was purified by FC (eluant n-hexane/ethyl acetate 3:1 (v/v))
O/
Scheme 1. Synthesis of Hydroxylated Benzoic Acid Amides 3
of Benzo[e][1.3]oxazin-2.4-dione 15
−7 and
3
to yield 1.45 g (30%) of colorless crystals. HPLC-MS (RP-18, H
acetonitrile 95:5 to 0:100 in 30 min and then 15 min isocratic,
2
+
APCI+): t
calcd for C15
CD OD): δ 7.62 (1H, dd, J ) 8.7 Hz, J ) 0.3 Hz, H-6), 6.86 (1H, d,
J ) 8.2 Hz, H-5′), 6.81 (1H, dd, J ) 2.2 Hz, J ) 0.3 Hz, H-2′), 6.77
1H, ddt, J ) 8.2 Hz, J ) 2.2 Hz, J ) 0.6 Hz, H-6′), 6.32 (1H, ddd,
J ) 8.7 Hz, J ) 2.4 Hz, J ) 0.3 Hz, H-5), 6.28 (1H, dd, J ) 2.4 Hz,
J ) 0.3 Hz, H-3), 4.43 (2H, bs, H-7′), 3.82 (3H, s, OCH
) ppm. 13
NMR (100 MHz; CD OD): δ 171.10 (C, C-7), 163.84 (C, C-2 or C-4),
63.58 (C, C-4 or C-2), 148.29 (C, C-4′), 147.66 (C, C-3′), 133.15 (C,
C-1′), 130.25 (CH, C-6), 119.86 (CH, C-6′), 115.68 (CH, C-2′), 112.82
R
15.54 min, m/z ) 289.89 (100%, [M + H] ). HRMS:
1
H
5
15NO 289.0950, found 289.0931. H NMR (400 MHz,
3
(
3
C
Table 1. Evaluation of Bitter-Masking Effect of Hydroxylated Benzoic
3
-
1
Acid Amides Compared to Homoeriodictyol 1 against 500 mg L
1
Caffeine
(
(
CH, C-5′), 108.78 (C, C-1), 108.47 (CH, C-5), 103.95 (CH, C-3), 56.49
CH , OCH ), 43.47 (CH , C-7′) ppm.
,7-Dihydroxy-3-(4-hydroxy-3-methoxybenzyl)benzo[e][1,3]oxazine-
,4-dione (15). Amide 9 (516 mg, 1.69 mmol) was dissolved in pyridine
bitter rating
3
3
2
panelists
(1−10)
5
compd all maskingb blank
test
profile (100 mg L- in 5% sucrose)
1
2
1c 10
10
10
7.4
5.0
nd
±
±
1.6 4.2
1.1 5.3
nd
±
±
1.7* sweet, vanillic, phenolic
1.2 fresh, fruity, sweet, mouthfeel
too bitter
0.9* vanillic, pepper, pungent, warming
0.5* fruity, sweet, dry−dusty
0.6* neutral, slightly vanillic, liquorice
1.0* cream, sweet, vanillic, caramel, toffee
0.9* sweet, vanillic, liquorice
(10 mL), and after cooling to 0 °C, benzyl chloroformate (1.2 mL,
3
.45 mmol) was added under nitrogen atmosphere using a syringe
3
4
5
6
7
8
9
0
16
during 10 min. The mixture was stirred at 0 °C for 2 h and between 0
and 20 °C for a further 12 h. After refluxing at 115 °C for 8 h, ice (50
g) was added and the crude product extracted with ethyl acetate. The
combined organic phases were washed with brine and saturated
NaHCO solution, dried with anhydrous Na SO , filtered, and evapo-
3 2 4
rated to dryness in vacuo. The crude product (1.3 g) was purified using
FC (eluant n-hexane/ethyl acetate 3:1 to 1:1) to yield 256 mg (46%)
of a pale yellow solid. HPLC-MS (RP-18, H
0
15
12
11
16
11
15
15
15
12
12
9
11
13
8
7
6
10
9
5.3
6.1
6.3
5.9
6.7
5.6
4.6
5.3
4.6
±
±
±
±
±
±
±
±
±
1.0 4.1
1.1 4.5
1.2 4.5
0.9 4.1
0.9 5.1
1.0 5.7
1.2 4.5
0.9 4.5
1.0 3.7
±
±
±
±
±
±
±
±
±
1
1.0 herbal, sweet, rotten
11
12
1.2 herbal, cinnamon, dry−dusty, cardboard
2
O/acetonitrile 80:20 to
1.2 fresh, cooling, sweet
13
0.8* vanillic, astringent, phenolic, dry
−dusty,
:100 in 30 min and then 15 min isocratic, APCI-): t
R
15.30 min,
-
-
long-lasting, weak tingling
0.8 herbal, bitter, phenolic, weak tingling
0.7 astringent, metallic, drying, resin
m/z ) 330.6 (100%, [M - H] ), 660.9 (0.4%, [2M - H] ) >95%.
1
14
16
16
8
7
4.4
5.8
±
±
1.1 3.9
1.0 5.1
±
±
HRMS: calcd for C16
MHz, CD OD): δ 7.06 (1H, d, J ) 2 Hz, H-2′), 6.91 (1H, ddt, J ) 8.1
Hz, J ) 2 Hz, J ) 0.5 Hz, H-6′), 6.73 (1H, J ) 8.1 Hz, H-5′), 6.18
1H, d, J ) 2.1 Hz, H-3 or H-5), 6.15 (1H, d, J ) 2.1 Hz, H-5 or H-3),
7
H13NO 331.0692, found 331.0702. H NMR (400
15
3
a
Test concentration 100 mg L- ; mean values and 95% confidence intervals
1
(
4
b
) ppm. 1 C NMR
3
are given; * means significant (p < 0.05); nd means not determined. Number of
panelists who rated the bitterness of test solution lower than standard solution.
.99 (2H, s, H-7′), 3.83 (3H, d, J ) 0.2 Hz, OCH
3
(
(
(
(
100 MHz; CD OD): δ 167.50 (C, C-7), 165.91 (C, C-2 or C-4), 163.30
3
c
As the sodium salt.
C, C-4 or C-2), 155.55 (C, C-6), 149.52 (C, carbamate-CO), 148.87
C, C-3′), 147.59 (C, C-4′), 128.89 (C, C-1′), 123.10 (CH, C-6′), 116.13
CH, C-5′), 113.97 (CH, C-2′), 100.29 (CH, C-5 or C-3), 95.25 (CH,
and the solution of the activated ester was directly added to the
amine or amine hydrochloride dissolved in an aqueous sodium
hydrogen carbonate solution (cf. Scheme 1). Amide 7 was
transferred into the sodium salt 8 by simple reaction with 1
equiv of sodium hydroxide and subsequent evaporation. The
sodium content of 8 corresponded to a monosodium salt (calcd
C-3 or C-5), 94.55 (CH, C-1), 56.13 (CH
ppm.
3 3 2
, OCH ), 45.74 (CH , C-7′)
Sensory Studies. For screening of taste-modulating effects, the test
compounds were added directly to an aqueous solution of the
appropriate bitter compound; occasionally, the mixture was treated for
several minutes in an ultrasonic bath to improve the dissolution process.
Panelists (healthy adults, no tasting problems known) were trained on
caffeine as bitter standard. Studies were performed in the morning hours
7
.40%, found 7.65%). In addition, the proton NMR of 8 is quite
similiar to that of 7; only the signals for H-3 and H-5 were
shifted to higher field and that of C-3 and C-5 as well as of
C-1 downfield. These shifts indicated the formation of a phenolic
anion in position 2 or 4 of the benzoic acid moiety; most
probably, a sodium complex was formed between the carbonyl
oxygen and the deprotonated hydroxy group at C-2. The cyclic
derivative 16 was synthesized starting from 2,4,6-trihydroxy-
benzoic acid N-(4-hydroxy-3-methoxybenzyl)amide (9) using
benzyl chloroformate in pyridine (cf. Scheme 1).
1
-2 h after breakfeast, during which time they were not allowed to
drink black or green tea or coffee due to adaptation to caffeine; only
one bitter test per day was performed. A minimum of 10 testers was
used in the descriptive test. The bitterness was rated on a scale of 1
(no taste) to 10 (very strong taste). For all experiments, the test solutions
were coded and in the case of color or cloudiness the cups were covered
using aluminum foil. Panelists were advised to test randomly mixed
samples in the given order by the sip and spit method. The raw sensory
data were analyzed using the standard functions of Microsoft Excel
All compounds were purified at least to 95% (HPLC) and
9
7. For calculation of significance, Student’s matched pair test was
1
13
used.
thoroughly characterized by H/ C NMR, HPLC-MS (APCI
mode), and for new compounds also by HRMS. The analytical
data obtained for aduncamide (3) and salicoyl tyramine (4) were
in accordance with the data published (14, 15).
RESULTS AND DISCUSSION
Syntheses. The polyhydroxylated amides were synthesized
using a protecting- group-free method described earlier for the
preparation of phenolic acid amides (19, 17) with moderate to
reasonably good yields as crystalline materials [average 40%,
between 18% for aduncamide (3) and 68% for salicylic acid
vanillylamide (5)]. The appropriate acid was esterified with N-hy-
droxysuccinimide using N,N′-dicyclohexylamide in 1,4-dioxane,
Screening for Bitter-Masking Activity. Prior to evaluation
as potential bitter masker, the flavor profile of each compound
-
1
was tested by an expert panel at 100 mg L in a 5% sucrose
solution. As shown in Table 1 all compounds show some flavor
but the intensities were generally very low and therefore not
rated. The influence of the weak intrinsic flavor on masking
tests was therefore neglected. The synthesized compounds were

8578 J. Agric. Food Chem., Vol. 54, No. 22, 2006
Ley et al.
Table 2. Bitter-Masking Effect of 100 mg L- 2,4-Dihydroxybenzoic
1
a
Acid Vanillylamide (7) against Various Bitter Compounds
bitter rating
(1−10)
%
reduction
of bitter
rating
panelists
masking
bitter compound
all
blank
test
-
1
caffeine, 500 mg L
11
16
16
16
11
12
12
8
6.3
±
±
±
±
1.2
1.3
1.1
1.0
4.5
4.1
5.1
6.5
±
±
±
±
0.6
29*
25*
21*
4
-1
quinine, 5 mg L
5.5
6.5
6.8
0.8
0.9
1.2
-
1
salicin, 250 mg L
H-Leu-Trp-OH,
-
1
2000 mg L
a
Test concentration 100 mg L-1; mean values and 95% confidence intervals
are given; * means significant (p < 0.05); nd means not determined.
To get more information about structural elements that may
be necessary or decrease the activity, we synthesized the amide
Figure 3. Masking effect of hydroxylated benzoic acid amides compared to
-
1
homoeriodictyol
1
1
against 500 mg
00 mg L ). Relative masking effect was calculated starting from averaged
bitter ratings of blank and test solution (cf. Table 1); * significant (p <
.05).
L
caffeine (test concentration
1
0 as the closest relative to eriodictyol (2) which was the most
-1
active bitter-masking flavanone in our previous study (47%
activity, 10). Surprisingly, the benzamide 10, which contains
the catechol moiety, showed no activity at all. On the other
hand, amide 11, which is the structural analogue to the more or
less inactive naringenin, was also not able to reduce the
bitterness of caffeine. Interestingly, the reducing power of the
phenethylamide 13, which is a homologue to 7, is only slightly
decreased. Amide 14, containing an isovanillyl unit, was
synthesized as an analogue of hesperetin. As in the case of the
flavanone (12% activity, 10), the masking activity was only
weak and not significant. Last but not least, we synthesized a
more constrained analogue of amide 9; the activity is largely
reduced but not fully eliminated.
0
screened for their bitter masking activity by comparison with a
-
1
5
00 mg L caffeine solution and a test solution containing the
caffeine and the test compound (cf. Table 1). To avoid
adaptation, the samples were anonymously coded and given to
the panelist in randomized order. Data from at least 10 panelists,
and in most cases of 15 or 16 panelists, were used. All ratings
were used for calculations, even in cases of possible adaptation
effects due to prior coffee or tea consumption. The bitterness
-
1
rating of a 500 mg L caffeine solution is constantly between
.4 and 6.7. The raw data were averaged for blank and for the
4
test solution, respectively, and the relative bitter inhibition was
calculated starting from the mean values (cf. Figure 3).
The naturally occurring aduncamide (3) showed no masking
effect, and salicoyl tyramine (4) showed an intrinsic bitterness. In
contrast to these discouraging results, the benzyl amides 5, 6,
Thus, the combination of free hydroxy groups in the benzoic
acid part which corresponds to ring A of flavanones and a
vanillyl unit in the amine moiety which corresponds to ring B
of flavanones seemed to be necessary for a masking activity
toward caffeine. In most cases, changes of the substitution
pattern at the amine part caused decrease or loss of activity, as
did methylations at the benzoic acid part.
Evaluation of 2,4-Dihydroxybenzoic Acid Vanillylamide
(7). The most promising candidate, 2,4-dihydroxybenzoic acid
N-(4-hydroxy-3-methoxybenzyl)amide (7), was evaluated more
in depth. First, the activity against other bitter components was
tested (cf. Table 2). As a prototypical bitter component quinine
was chosen; salicin was tested because the bitter receptor for this
class of compounds is now known (5). In addition, N-L-leucyl-
L-tryptophan (H-Leu-Trp-OH) was evaluated as a typical bitter
peptide; such hydrophobic peptides can cause a lot of bitter taste
problems in processed food applications (20). Similiar to the
results for homoeriodictyol (10), amide 7 was able to reduce the
perceived bitterness of quinine and salicin by about 20% but
was not active toward H-Leu-Trp-OH. Additionally, the dose-
7
, and 9, which are much closer structural relatives to homo-
eriodictyol (1) (they contain the vanillyl moiety and at least
parts of the substitution pattern of flavanone ring A) were able
to reduce significantly the bitterness of caffeine by 20-30%.
As a result, it seemed to be possible to reduce the complexity
of the parent bitter masker without loss of activity. When parent
compound 7 was methylated at the benzoic part to yield
vanillylamide 12, which resembled the moderate bitter masker
sterubin (eriodictyol-7-methyl ether, 20% activity, 10), the
activity to inhibit the bitterness of caffeine was slightly reduced.
In our previous study (10) we had found that the monosodium
salt of homoeriodictyol was more active (43%) than the parent
flavanone (28%). Thus, we prepared the corresponding sodium
salt 8 of the most active vanillylamide 7. In contrast to
homoeriodictyol, the activity is only slightly increased.
Figure 4. Dose
semilogarithmic plot.
−
response plot of masking effect of 2,4-dihydroxybenzoic acid vanillylamide (7) in 500 mg L-1 caffeine solution. (A) linear plot and (B)

Hydroxylated Benzoic Acid Amides as Bitter-Masking Compounds
J. Agric. Food Chem., Vol. 54, No. 22, 2006 8579
(
4) Behrens, M.; Brockhoff, A.; Kuhn, C.; Bufe, B.; Winnig, M.;
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(
(
(
5) Bufe, B.; Hofmann, T.; Krautwurst, D.; Raguse, J.-D.; Meyerhof,
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Figure 5. Masking effect of a combination of homoeriodictyol (1) and
-
1
vanillylamide (7) toward 500 mg L
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(
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dependent activity of 7 toward the bitterness of a 500 mg L-1
caffeine solution was determined. The results (cf. Figure 4A)
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-
1
at 500 mg L and a more or less linear relationship between 5
(9) Gravina, S. A.; McGregor, R. A.; Nossoughi, R.; Kherlopian,
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1
and 500 mg L when plotted semilogarithmically (cf. Figure
B). As a further experiment, the activity of a 1:1 mixture of
4
homoeridictyol (1) and amide 7 was compared with the values
obtained for the single compounds. As shown in Figure 5, there
was only a more or less additive effect, and no significant
synergism or antagonism could be found. These results suggest
that both compounds act as competitors on the molecular level.
In conclusion, we have found structural analogues to bitter-
masking flavanones that also were able to reduce the bitterness
of caffeine solutions. The most interesting compounds were the
hydroxylated benzoic acid vanillylamides 5-9. The compounds,
which have only a weak flavor profile, show activities compa-
rable to homoeriodictyol (1). All further variations in structure
caused a decrease or loss in bitter-masking ability. Summarizing
the data for the most active amide 7, we conclude that the
hitherto unknown mechanism of inhibition of flavanones and
the new amides is probably the same, because the behavior of
both structural classes was nearly the same. In the future, the
new amides may be alternatives for the expensive flavanones
to create flavor solutions to mask bitterness of pharmaceuticals
or foodstuffs. Besides these possible applications, the new
structures may be used as a tool to find the molecular mechanism
responsible for the bitter masking effect.
(
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(
(
(
ABBREVIATIONS USED
AMP, adenosine monophosphate; APCI, atmospheric pressure
chemical ionization; FC, flash chromatography; H-Leu-Trp-OH,
N-L-leucyl-L-tryptophan; tR, retention time.
2
003, 19, 197-202.
(
(
(
(
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ACKNOWLEDGMENT
18) Ley, J. P. Phenolic acid amides of phenolic benzylamines against
We thank C.-O. Schmidt, M. Roloff, and B. Weber for the
analytical services.
UVA-induced oxidative stress in skin. Int. J. Cosmet. Sci. 2001,
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3, 35-48.
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Received for review June 19, 2006. Revised manuscript received August
0, 2006. Accepted September 3, 2006.
3
JF0617061