- Email: [email protected]
Structure-activity relationships of trigeminal effects for artificial and naturally occurring alkamides related to spilanthol
W.L.P. Bredie and M.A. Petersen (Editors) Flavour Science: Recent Advances and Trends 9 2006 Elsevier B.V. All rights reserved.
21
Structure-activity relationships of trigeminal effects for artificial and naturally occurring alkamides related to spilanthol Jakob P. Ley, Gerhard Krammer, Jan Looft, Gerald Reinders and Heinz-Jiirgen Bertram Corporate Research & Development, Symrise GmbH & Co. KG, P.O. Box 1253, 37601 Holzminden, Germany
ABSTRACT Twenty six variations of the structure of spilanthol ((E,E,Z)-2,6,8-decatrienoic acid Nisobutylamide) were synthesised starting from the appropriate unsaturated acids by amidation with simple aliphatic amines. The resulting alkamides were evaluated for their trigeminal sensory properties (burning, pungency, tingling, scratching, numbing, warming, mouth-watering, cooling). Spilanthol was the most active tingling and mouthwatering compound, trans-Pellitorine ((E,E)-2,4-decadienoic acid N-isobutylamide) showed mainly the same mouth-watering activity without the strong tingling effect. All other structural variations led to lower intensity of the trigeminal activity. 1. I N T R O D U C T I O N The alkamide spilanthol ((E,E,Z)-2,6,8-decatrienoic acid N-isobutylamide), which occurs in several plants of the Compositae family, e.g. in Spilanthes acmella, shows a pronounced tingling and mouth-watering effect on ingestion [1]. For shogaols (2hydroxyisobutylamides of various unsaturated fatty acids) and some other alkamides, the structure-activity of the tingling effect has been described earlier [2,3]. In a further study it was shown that the stereochemistry of the fatty acid part strongly influences the sensory properties [4]. To our knowledge, no study exists covering all important trigeminal effects on natural and synthesised alkamides.
22 2. MATERIALS AND METHODS
2.1. Isolation and syntheses Spilanthol (1) and homospilanthol (11) were isolated from Spilanthes acmella concentrated extract (Jambu oleoresin, Robertet Flavors) by preparative HPLC. Most other amides were synthesised by amidation of the appropriate acid chloride with an amine, cis-Pellitorine (9) and derivatives 16 and 17 were synthesised by an enzymatic conversion of pear ester according to the literature [4]. The acid for dihydrospilanthol (2) was synthesised by a Wittig-sequence starting from acrolein and 6-bromohexanoic acid and subsequent treatment with oxalyl chloride. The acid for amide 3 was prepared by a Wittig-Horner reaction of (Z)-5-octenal with triethylphosphonoacetate with subsequent saponification in KOH. (E,E)-2,4-Decadienoic acid, (E)-2-decen-4-ynoic acid and (E,E)-2,4-undecadienoic acid were synthesised by the same procedure starting from (E)-2-octenal, 2-octinal and (E)-2-nonenal, respectively. (E)-2-Decenoic acid and (E)-3-nonenoic acid were prepared from the commercial available ethyl esters by saponification. The amides 1, 2, 4, 6, 7, 9, 11, 12, 14, 18, 19, 20, and 21 were found in nature [2,3,5].
2.1.1. Amidation In a typical amidation reaction 10 g (50 mmol) of (E)-2-decenoic acid chloride (prepared from the free acid with SOC12) were dissolved in 10 ml acetone and added to a solution of 60 mmol amine in 10 ml acetone and 25 ml NaOH (9.64 g NaOH/100 ml water). In most cases a crystalline product could be obtained. If the amide separated as an oily product, a further chromatographic clean-up was performed. All amides were purified to >95% purity (GC) and fully characterised by NMR, MS and in the case of new compounds by HRMS. 2.2. Sensory testing The test compound was dissolved in ethanol (30 or l0 mg/5 ml) and 1 ml of the solution further diluted with 11% sucrose in water (200 ml). For evaluation, the test solutions (2 to 5 ml) were sipped for 10 to 20 s and then spat out. Tasting sessions were performed by 6 to 8 fully informed and trained panellists. No more than 1 or 2 test samples were evaluated per day. Estimated intensity ratings for descriptors were 1 (low) to 9 (high). 3. RESULTS Most of the 26 evaluated alkamides (Tables 1 and 2) showed at least a moderate trigeminal sensation, especially the tingling effect. Spilanthol (1) was the most active tingling and mouth-watering compound, trans-Pellitorine (7) showed mainly the same mouth-watering activity without the strong tingling effect of 1. (E)-2-Decen-4-ynoic acid N-isobutylamide (dehydropellitorine, 10) caused a moderate tingling and mouthwatering effect. (E)-2-Decenoic acid N-isobutylamide (dihydropellitorine, 4) induced saliva flow but showed only a very weak tingling effect. The mouth-watering effect was
23 strongly affected by small variations of the amide moiety as well as by the fatty acid residue, cis-Pellitorine ((E,Z)-2,4-decadienoic acid N-isobutylamide, 9) showed high pungency and a warming effect, but no mouth-watering and tingling at all. Table 1. Sensory profiles of N-isobutylamides and other alkamides in 11% sucrose solution.
Structure
Name
Conc. E ~ (ppm) ~ m E
~ ~
~ ~o
0
Spilanthol
30
8
Dihydrospilanthol
10
2
7
0
0
3 -...'='-..-...~tN~ 0
4 ~ N ~ U 0
5 ~ H ' U 0
6 ~ N ' ~
(E,Z)-2,7-Decadienoic acid N-isobutylamide (E)-2-Decadienoic acid N-isobutylamide (E)-3-Nonadienoic acid N-isobutylamide Decanoic acid Nisobutylamide
10
n. n. n. n. n. n. n. n. a.
10
a.
a.
a.
a.
2
a.
a.
a.
4 5
10
3
10
2
trans-Pellitorine
10
2 5
(E,E)-2,4-Undecadienoic acid N-isobutylamide
10
Cis-Pellitorine
10
Dehydropellitorine
10
4
4
Homospilanthol
30
5
5
(E)-2-Decenoic acid N-(2-methylbutyl)amide
10
Dehydrohomopellitorine
10
4
Homopellitorine
10
2 3
(2'S)-Homopellitorine
10
4
Cis-Homopellitorine
10
( 2 'S)-cis-H omope llitorine
10
0
7
~
N
~
(
0
8 ~ N " U
4
7
3
0
5
5
0
4 4
O
11 ~
N
12 ~
~
~
" 0
"
-
~
3 3
3
O
3
4 4
O
14 ~
N
~
" O
15
~
H
N
H
~'~'-
n.n. a.
a.
n.a.: not available9
=
3
n.
n.
n.
n.
n.
n.
a.
a.
a.
a.
a
a.
o H
4
3
24 Table 2. Sensory profiles of further alkamides in 11% sucrose solution.
..= ~ :~ ~ . - " Structure
Name
.= ..=
Conc. (ppm)
O
19 ~
Dihydroacchilleamide
10
Achilleamide
10
3 5
Sarmentine
10
3
(E)-2-Decenoyl N-pyrrolidine (E,E)-2,4-Decadienoic acid N- l'-methylpropylamide (E,E)-2,4-Decadienoic acid N-butylamide (E,E)-2,4-Decadienoic acid N-(3-Methylbutyl)amide (E,E)-2,4-Decadienoic acid N-(2-hydroxyethyl)amide (E,E)-2,4-Decadienoic acid N-(2-ethylhexyl)amide
10
334
3
O
~
O
O
O
22 ~
~-'1"-I O
23 ~
N
H
~/~
O
24 ~ 25 ~
N N
H
~
O
O
~'j'-
H H
O
26 ~ ~ " ~
4
10 10
2
3
3
10
4
10
4
4
4
10
4
4
2 3
3
4. DISCUSSIONS AND C O N C L U S I O N The t r a n s 2 double bond is necessary for the mouth-watering property, as stated earlier by Galoph et al. [2] and as exemplified in our study by variation I to 2. The change of diene to monoene moiety (1 to 3) resulted in a loss of this physiologic activity. Variations in the double bond pattern between C2 and C5 (4 to 10) yielded interesting trigeminal activities, but the most active compounds besides spilanthol (1) were the 2,4di-unsaturated amides 7, 9 and 10. Changing the amine moiety always causes a decrease and sometimes a change in the pattern of trigeminal activity compared to the appropriate N-isobutylamide. References
1. R.S. Ramsewak, A.J. Erickson and M.G. Nair, Phytochem., 51 (1999) 729. 2. T. Hofmann, C.-H. Ho and W. Pickenhagen (eds.), Challenges in taste chemistry and biology, Washington, DC, USA (2003) 139. 3. P. Given and D. Paredes (eds.), Chemistry of taste, Washington, DC, USA (2002) 202. 4. J.P. Ley, J.-M. Hilmer, B. Weber, G. Krammer, I.L. Gatfield and H.-J. Bertram, Eur. J. Org. Chem., 24 (2004) 5135. 5. G.M. Strunz, Stud. Nat. Prod. Chem., 24 (2000) 683.
21
Structure-activity relationships of trigeminal effects for artificial and naturally occurring alkamides related to spilanthol Jakob P. Ley, Gerhard Krammer, Jan Looft, Gerald Reinders and Heinz-Jiirgen Bertram Corporate Research & Development, Symrise GmbH & Co. KG, P.O. Box 1253, 37601 Holzminden, Germany
ABSTRACT Twenty six variations of the structure of spilanthol ((E,E,Z)-2,6,8-decatrienoic acid Nisobutylamide) were synthesised starting from the appropriate unsaturated acids by amidation with simple aliphatic amines. The resulting alkamides were evaluated for their trigeminal sensory properties (burning, pungency, tingling, scratching, numbing, warming, mouth-watering, cooling). Spilanthol was the most active tingling and mouthwatering compound, trans-Pellitorine ((E,E)-2,4-decadienoic acid N-isobutylamide) showed mainly the same mouth-watering activity without the strong tingling effect. All other structural variations led to lower intensity of the trigeminal activity. 1. I N T R O D U C T I O N The alkamide spilanthol ((E,E,Z)-2,6,8-decatrienoic acid N-isobutylamide), which occurs in several plants of the Compositae family, e.g. in Spilanthes acmella, shows a pronounced tingling and mouth-watering effect on ingestion [1]. For shogaols (2hydroxyisobutylamides of various unsaturated fatty acids) and some other alkamides, the structure-activity of the tingling effect has been described earlier [2,3]. In a further study it was shown that the stereochemistry of the fatty acid part strongly influences the sensory properties [4]. To our knowledge, no study exists covering all important trigeminal effects on natural and synthesised alkamides.
22 2. MATERIALS AND METHODS
2.1. Isolation and syntheses Spilanthol (1) and homospilanthol (11) were isolated from Spilanthes acmella concentrated extract (Jambu oleoresin, Robertet Flavors) by preparative HPLC. Most other amides were synthesised by amidation of the appropriate acid chloride with an amine, cis-Pellitorine (9) and derivatives 16 and 17 were synthesised by an enzymatic conversion of pear ester according to the literature [4]. The acid for dihydrospilanthol (2) was synthesised by a Wittig-sequence starting from acrolein and 6-bromohexanoic acid and subsequent treatment with oxalyl chloride. The acid for amide 3 was prepared by a Wittig-Horner reaction of (Z)-5-octenal with triethylphosphonoacetate with subsequent saponification in KOH. (E,E)-2,4-Decadienoic acid, (E)-2-decen-4-ynoic acid and (E,E)-2,4-undecadienoic acid were synthesised by the same procedure starting from (E)-2-octenal, 2-octinal and (E)-2-nonenal, respectively. (E)-2-Decenoic acid and (E)-3-nonenoic acid were prepared from the commercial available ethyl esters by saponification. The amides 1, 2, 4, 6, 7, 9, 11, 12, 14, 18, 19, 20, and 21 were found in nature [2,3,5].
2.1.1. Amidation In a typical amidation reaction 10 g (50 mmol) of (E)-2-decenoic acid chloride (prepared from the free acid with SOC12) were dissolved in 10 ml acetone and added to a solution of 60 mmol amine in 10 ml acetone and 25 ml NaOH (9.64 g NaOH/100 ml water). In most cases a crystalline product could be obtained. If the amide separated as an oily product, a further chromatographic clean-up was performed. All amides were purified to >95% purity (GC) and fully characterised by NMR, MS and in the case of new compounds by HRMS. 2.2. Sensory testing The test compound was dissolved in ethanol (30 or l0 mg/5 ml) and 1 ml of the solution further diluted with 11% sucrose in water (200 ml). For evaluation, the test solutions (2 to 5 ml) were sipped for 10 to 20 s and then spat out. Tasting sessions were performed by 6 to 8 fully informed and trained panellists. No more than 1 or 2 test samples were evaluated per day. Estimated intensity ratings for descriptors were 1 (low) to 9 (high). 3. RESULTS Most of the 26 evaluated alkamides (Tables 1 and 2) showed at least a moderate trigeminal sensation, especially the tingling effect. Spilanthol (1) was the most active tingling and mouth-watering compound, trans-Pellitorine (7) showed mainly the same mouth-watering activity without the strong tingling effect of 1. (E)-2-Decen-4-ynoic acid N-isobutylamide (dehydropellitorine, 10) caused a moderate tingling and mouthwatering effect. (E)-2-Decenoic acid N-isobutylamide (dihydropellitorine, 4) induced saliva flow but showed only a very weak tingling effect. The mouth-watering effect was
23 strongly affected by small variations of the amide moiety as well as by the fatty acid residue, cis-Pellitorine ((E,Z)-2,4-decadienoic acid N-isobutylamide, 9) showed high pungency and a warming effect, but no mouth-watering and tingling at all. Table 1. Sensory profiles of N-isobutylamides and other alkamides in 11% sucrose solution.
Structure
Name
Conc. E ~ (ppm) ~ m E
~ ~
~ ~o
0
Spilanthol
30
8
Dihydrospilanthol
10
2
7
0
0
3 -...'='-..-...~tN~ 0
4 ~ N ~ U 0
5 ~ H ' U 0
6 ~ N ' ~
(E,Z)-2,7-Decadienoic acid N-isobutylamide (E)-2-Decadienoic acid N-isobutylamide (E)-3-Nonadienoic acid N-isobutylamide Decanoic acid Nisobutylamide
10
n. n. n. n. n. n. n. n. a.
10
a.
a.
a.
a.
2
a.
a.
a.
4 5
10
3
10
2
trans-Pellitorine
10
2 5
(E,E)-2,4-Undecadienoic acid N-isobutylamide
10
Cis-Pellitorine
10
Dehydropellitorine
10
4
4
Homospilanthol
30
5
5
(E)-2-Decenoic acid N-(2-methylbutyl)amide
10
Dehydrohomopellitorine
10
4
Homopellitorine
10
2 3
(2'S)-Homopellitorine
10
4
Cis-Homopellitorine
10
( 2 'S)-cis-H omope llitorine
10
0
7
~
N
~
(
0
8 ~ N " U
4
7
3
0
5
5
0
4 4
O
11 ~
N
12 ~
~
~
" 0
"
-
~
3 3
3
O
3
4 4
O
14 ~
N
~
" O
15
~
H
N
H
~'~'-
n.n. a.
a.
n.a.: not available9
=
3
n.
n.
n.
n.
n.
n.
a.
a.
a.
a.
a
a.
o H
4
3
24 Table 2. Sensory profiles of further alkamides in 11% sucrose solution.
..= ~ :~ ~ . - " Structure
Name
.= ..=
Conc. (ppm)
O
19 ~
Dihydroacchilleamide
10
Achilleamide
10
3 5
Sarmentine
10
3
(E)-2-Decenoyl N-pyrrolidine (E,E)-2,4-Decadienoic acid N- l'-methylpropylamide (E,E)-2,4-Decadienoic acid N-butylamide (E,E)-2,4-Decadienoic acid N-(3-Methylbutyl)amide (E,E)-2,4-Decadienoic acid N-(2-hydroxyethyl)amide (E,E)-2,4-Decadienoic acid N-(2-ethylhexyl)amide
10
334
3
O
~
O
O
O
22 ~
~-'1"-I O
23 ~
N
H
~/~
O
24 ~ 25 ~
N N
H
~
O
O
~'j'-
H H
O
26 ~ ~ " ~
4
10 10
2
3
3
10
4
10
4
4
4
10
4
4
2 3
3
4. DISCUSSIONS AND C O N C L U S I O N The t r a n s 2 double bond is necessary for the mouth-watering property, as stated earlier by Galoph et al. [2] and as exemplified in our study by variation I to 2. The change of diene to monoene moiety (1 to 3) resulted in a loss of this physiologic activity. Variations in the double bond pattern between C2 and C5 (4 to 10) yielded interesting trigeminal activities, but the most active compounds besides spilanthol (1) were the 2,4di-unsaturated amides 7, 9 and 10. Changing the amine moiety always causes a decrease and sometimes a change in the pattern of trigeminal activity compared to the appropriate N-isobutylamide. References
1. R.S. Ramsewak, A.J. Erickson and M.G. Nair, Phytochem., 51 (1999) 729. 2. T. Hofmann, C.-H. Ho and W. Pickenhagen (eds.), Challenges in taste chemistry and biology, Washington, DC, USA (2003) 139. 3. P. Given and D. Paredes (eds.), Chemistry of taste, Washington, DC, USA (2002) 202. 4. J.P. Ley, J.-M. Hilmer, B. Weber, G. Krammer, I.L. Gatfield and H.-J. Bertram, Eur. J. Org. Chem., 24 (2004) 5135. 5. G.M. Strunz, Stud. Nat. Prod. Chem., 24 (2000) 683.