Application of hansch's model to capsaicinoids and capsinoids: A study using the quantitative structure-activity relationship. A novel method for the synthesis of capsinoids

Article abstract of DOI:10.1021/jf9035029

We describe a synthetic approach for two families of compounds, the capsaicinoids and capsinoids, as part of a study of the quantitative relationship between structure and activity. A total of 14 capsaicinoids of increasing lateral chain lengths, from 2 to 16 carbon atoms, were synthesized. In addition, 14 capsinoids with identical lateral chains, as well as capsiate and dihydrocapsiate, have been synthesized, and a new method for the synthesis of these compounds has been developed. The yields range from 48.35 to 98.98%. It has been found that the synthetic capsaicinoids and capsinoids present a lipophilia similar to those of the natural compounds and present similar biological activity. The bioactivity of the synthetic capsaicinoids and capsinoids decreases proportionally to the degree of difference in lipophilia (higher or lower) compared to the natural compounds. Biological activity was determined using the etiolated wheat (Triticum aestlvum L.) coleoptiles bioassay and by comparing results of the synthesis with those presented by their counterpart natural compounds. The bioactivities found correlated directly to the lipophilic properties of the synthesized compounds.

Full text of DOI:10.1021/jf9035029

3342 J. Agric. Food Chem. 2010, 58, 3342–3349
DOI:10.1021/jf9035029
Application of Hansch’s Model to Capsaicinoids and
Capsinoids: A Study Using the Quantitative Structure-Activity
Relationship. A Novel Method for the Synthesis of Capsinoids
ERARDO F. BARBERO,† JOSE M. G. MOLINILLO,‡ ROSA M. VARELA,‡ MIGUEL
P
ALMA,*,†
´
G
‡
†
´
F
RANCISCO A. MACIAS
,
AND
CARMELO G. BARROSO
†
‡
ꢀ
Department of Analytical Chemistry and Department of Organic Chemistry, University of Cadiz,
ꢀ
ꢀ
Avda. Republica Saharaui, s/n, 11510 Puerto Real, Cadiz, Spain
We describe a synthetic approach for two families of compounds, the capsaicinoids and capsinoids,
as part of a study of the quantitative relationship between structure and activity. A total of 14
capsaicinoids of increasing lateral chain lengths, from 2 to 16 carbon atoms, were synthesized. In
addition, 14 capsinoids with identical lateral chains, as well as capsiate and dihydrocapsiate, have
been synthesized, and a new method for the synthesis of these compounds has been developed. The
yields range from 48.35 to 98.98%. It has been found that the synthetic capsaicinoids and capsinoids
present a lipophilia similar to those of the natural compounds and present similar biological activity.
The bioactivity of the synthetic capsaicinoids and capsinoids decreases proportionally to the degree of
difference in lipophilia (higher or lower) compared to the natural compounds. Biological activity was
determined using the etiolated wheat (Triticum aestivum L.) coleoptiles bioassay and by comparing
results of the synthesis with those presented by their counterpart natural compounds. The bioactivities
found correlated directly to the lipophilic properties of the synthesized compounds.
KEYWORDS: Capsinoids; capsaicinoids; synthesis of capsaicinoid and capsinoid; lipophilia; QSAR;
Hansch’s transport model, Lipinski’s rule of five; log P; bioassays; IALOGP
INTRODUCTION
[4-hydroxy-3-methoxybenzyl-7-methyloctanoate], have also been
isolated. The acyl residues of capsiate, dihydrocapsiate, and
nordihydrocapsiate are the same as those of capsaicin, dihydro-
capsaicin, and nordihydrocapsaicin, respectively (15).
Capsaicinoids (1) and capsinoids (2) are two families of
compounds present in pepper (Capsicum annuum L.) that are
intimately related. Capsaicinoids are responsible for the hot, spicy
flavor of peppers (3). Chemically, they are a group of acid amides
formed from vanillyl amine and fatty acids of 8-13 carbon
atoms. Capsaicin is (E)-N-[(4-hydroxy-3-methoxyphenyl)methyl]-
8-methylnon-6-enamide, and dihydrocapsaicin is the 6,7-dihydro
derivative of capsaicin. They are notable among the various
natural capsaicinoids. These two major capsaicinoids generally
account for 90% of the total capsaicinoids in the spicy varieties of
peppers (4, 5). In addition to these two major compounds, more
than 18 different capsaicinoids have been found as minor com-
pounds (6, 7). Capsaicinoids present antimutagenic and antitu-
mor properties (8, 9), are potent antioxidants (10), are employed
as topical analgesics for treating pain (11), and possess anti-
inflammatory (12) and antimicrobial (13) properties.
Capsinoids have biological properties similar to those of
capsaicinoids, except that they are not hot or spicy. These
compounds have several notable biological properties: they are
powerful antioxidants (16) and possess chemopreventive and
antineoplastic properties (17); they promote energy consumption
and suppress the accumulation of fats in the organism (18); they
increase body temperature and the consumption of oxygen in
humans (19), and present powerful anti-inflammatory acti-
vity (20). These applications and biological effects of the capsai-
cinoids, together with their lack of spicy properties, make them
extremely important, since their application in both food and
pharmacological preparations is not as limited as the capsaici-
noids, due to the pungency of the latter.
Many authors have studied the chemical synthesis of capsai-
cinoids (21,22), and procedures for the preparation and purifica-
tion of synthetic capsaicin have been patented (23). However,
because capsinoids were discovered more recently than their
analogues (2, 14), procedures for their synthesis have lagged
behind. However, some studies dealing with their synthesis
have been published. Kobata et al. reported the chemical synth-
esis of dihydrocapsiate (2), and later synthesized other ana-
logues such as vanillyl nonanoate (24). Appendino et al. (25)
studied the chemoselective esterification of phenolic acids with
alcohols, and thereby synthesized vanillyl nonanoate. Recently,
Capsinoids are structurally similar to capsaicinoids, except for
the way that the carbon chain is bound to the aromatic ring: an
amide moiety in capsaicinoids and an ester moiety in capsinoids.
Capsinoids have been isolated from the fruit of a sweet pepper
cultivar, CH-19 Sweet (2, 14). The most abundant natural capsi-
noid is capsiate [4-hydroxy-3-methoxybenzyl (E)-8-methyl-6-
nonenoate]. Other capsinoids, such as dihydrocapsiate [4-hydroxy-
3-methoxybenzyl-8-methylnonanoate] and nordihydrocapsiate
*To whom correspondence should be addressed. Phone: þ34-
956016360. Fax þ34-956016460. E-mail: miguel.palma@uca.es.
pubs.acs.org/JAFC
Published on Web 02/24/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3343
Figure 1. Acylation of 4-hydroxy-3-methoxy benzylamine hydrochloride (1) with acyl chlorides (2a-n).
Torregiani et al. (26) developed a new method for the selective
esterification of phenolic alcohols, catalyzing the reaction with
cerium(III) chloride.
Here we present an easy and selective method for the synthesis
of capsinoids. This has been used to synthesize two natural capsi-
noids (capsiate and dihydrocapsiate) and synthetic analogues
of varying lateral chain length, to study the structure-activity
relationship.
Lipophilia, which measures the ability of a compound to cross
cell membranes, is a key factor in the absorption of any bioactive
compound, as it determines its bioavailability in the cell. This is
usually expressed through the logarithm of the octanol-water
partition coefficient (log P) (27, 28) and is present in many
equations of quantitative structure-activity relationship (QSAR),
because an optimum equilibrium must exist between water (the
usual carrier), solubility, and lipophilia.
The partition coefficient is widely used in pharmacology and
pharmacognosy studies, and has recently been highlighted in the
so-called “Lipinski’s rule of five” (29). Using computational
methods to study those factors affecting the activity, the rule
predicts poor absorption and permeation for those compounds
having more than 5 H-bond donors, 10 H-bond acceptors, a mole-
cular mass greater than 500 Da, and a calculated log P greater
than 5. Those compounds whose characteristics fall outside the
limits of this rule are supposed to be either completely inactive or
only poorly active.
hydrogen carbonate (99.0-100.5%), sodium hydroxide (98.0-100.5%),
methanol (HPLC-grade), N,N-dimethylmethanamide (DMF) (99%), de-
hydrated pyridine (99%), and tetrahydrofuran (THF) (99.5%) were
´ ´
purchased from Panreac Quımica S.A. (Castellar del Valles, Barcelona,
Spain). Undecanoic acid (99%) (4j) and 8-methylnonanoic acid (97%)
were purchased from Acros Organics (New Jersey, USA). cis-8-Methyl-
6-nonenoic acid (97%) (10) was purchased from Maybridge (Tintagel,
Cornwall, UK). Ethyl acetate, chloroform, stabilized with ethanol, and
hexane were purchased from Scharlau Chemie S.A. (Sentmenat, Barcelona,
Spain). Thionyl chloride (99%) was purchased from Merck (Hohenbrunn,
Germany). Sodium nitrite (99%) was purchased from Merck (Darmstadt,
Germany). 4-Hydroxy-3-methoxybenzaldehyde (vanillin) (5) was purcha-
sed from Fluka AG, Buchs SG (Switzerland).
General Procedure for the Synthesis of Capsaicinoids. A modifi-
cation of the method developed by McIlvain et al. (23) was employed for
the synthesis of capsaicinoids.
Acylation of 4-Hydroxy-3-methoxy Benzylamine Hydrochloride (1)
with Acyl Chlorides (2a-n). To obtain the various capsaicinoids (3a-n),
acylations of 4-hydroxy-3-methoxy benzylamine hydrochloride (1) were
performed with the corresponding acyl chlorides (2a-n), according to the
particular capsaicinoid to be synthesized (Figure 1).
A small amount of 4-hydroxy-3-methoxy benzylamine hydrochloride
(1) (0.5-1 g) was placed in a 250 mL round-bottom flask and dissolved in
15 mL of DMF. An atmosphere of argon was introduced, and the mixture
was stirred until all of the initial compound was dissolved. Subsequently,
1 mL of 5 N NaOH was slowly added, dropwise, in an ice bath. Once the
NaOH has been added, the ice bath was removed and the mixture was left
in agitation at ambient temperature for 60 min. Then, the particular acyl
chloride (2a-n) of the required carbonated chain (2.5 equiv), dissolved in
2 mL of dry THF, was slowly added, using a syringe, over an ice bath.
Once the appropriate acyl chloride was added, the reaction mixture was
magnetically stirred and under argon at ambient temperature for 18 h. The
reaction was followed by TLC (eluent: 1% of methanol in chloroform;
chromogenic reagent: oleum). Then, the reaction was stopped by addition
of water. After purification of the reaction mixture, the various capsaici-
noids were obtained. The products formed, and the reaction yields, are
shown in Table 1.
To detect the bioactivity of these compounds, a bioassay invol-
ving elongation of etiolated wheat (Triticum aestivum L.) coleop-
tiles was used. This bioassay was originally devised for evalua-
ting the activity of auxins as plant growth regulators (30, 31),
although it has been more recently used to detect pharmacologi-
cal activities in chemical products, a purpose for which the
bioassay has been widely utilized and approved (32, 33).
Therein, the general bioactivity values have been determined,
and, finally, an examination to establish a correlation between the
degree of lipophilia and bioactivity has been carried out.
Formation of Acyl Chlorides (2j,l) from their Corresponding Acids (4j,l).
Two acids (undecanoic acid (4j) and tridecanoic acid (4l)) were trans-
formed into their corresponding acyl chlorides (2j,l), and the acylations of
4-hydroxy-3-methoxy benzylamine hydrochloride (1) were performed
later.
MATERIALS AND METHODS
The acids were placed in a 50 mL round-bottom flask, and argon was
introduced. A quantity of thionyl chloride, sufficient to dissolve the acid,
was added very slowly, dropwise, from a syringe, and stirred magnetically,
following addition of thionyl chloride, the reaction mixture was heated at
60 °C for 1 h. Subsequently, the excess thionyl chloride was eliminated in
vacuo, and a transparent oil was obtained. The acyl chloride formed was
dissolved in a known quantity of dry THF to be utilized in the subsequent
acylations of 4-hydroxy-3-methoxy benzylamine hydrochloride (1).
General Procedure for the Synthesis of Capsinoids. This new
method for synthesis comprises four stages. The two first stages are common
to all the capsinoids, but the last two stages are different for eachcapsinoid,
since they depend on the chain length required.
General Experimental Procedures. The purity of each compound
was determined by 1H NMR and 13C NMR analyses, and was found to be
g98%. 1H and 13C spectra were recorded using CDCl3 as the solvent, in a
Varian INOVA spectrometer, at 399.952 and 100.577 MHz, respectively.
The resonances of residual chloroform for 1Hand13CweresettoδH 7.25 ppm
and δC 77.00 ppm, respectively, and used as internal reference. UV-vis
spectra were obtained using a Varian Cary 50 BIO spectrophotometer,
with chloroform as the solvent.
Starting Materials. 4-Hydroxy-3-methoxy benzylamine hydrochlo-
ride (98%) (1), propionyl chloride (98%) (2b), butyryl chloride (98%) (2c),
pentanoyl chloride (98%) (2d), hexanoyl chloride (99%) (2e), heptanoyl
chloride (99%) (2f), octanoyl chloride (99%) (2g), nonanoyl chloride
(96%) (2h), decanoyl chloride (98%) (2i), lauroyl chloride (98%) (2k),
tridecanoic acid (4l), myristoyl chloride (97%) (2m), palmitoyl chloride
(98%) (2n), dihydrocapsaicin (90%) (3o), capsaicin (97%) (3p), t-butyl-
dimethyl silyl chloride (97%), and di-isobutyl aluminum hydride (1 M in
toluene) (DIBAL) were purchased from Sigma-Aldrich Chemie (Steinheim,
Germany). Acetic anhydride (98%) (2a), hydrochloric acid (37%), nitric
acid (65%), sodium chloride (99.0-100.5%), ethanol (99.5%), sodium
Silylation of Vanillin (5). The product was taken as the starting point
for the synthesis of capsinoids was vanillin (5) (4-hydroxy-3-meth-
oxybenzaldehyde). The vanillin (5) (5.900 g, 0.038 mol) was placed in a
250 mL round-bottom flask, and dissolved in about 30 mL of dry pyridine.
Then 1.2 equiv of t-butyldimethyl silyl chloride (TBDMSCl) (7.012 g,
0.046 mol) was added to this solution. The reaction mixture was stirred
and under argon at ambient temperature for 24 h. The reaction was

3344 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Table 1. Capsaicinoids and Yields Obtained
Barbero et al.
Esterification of 4-t-Butyldimethylsilyloxy-3-methoxybenzyl Alcohol
(7) with Acyl Chloride (2a-p). The next reaction (Figure 5) was the
esterification of 4-t-butyldimethylsilyloxy-3-methoxybenzyl alcohol (7)
with acyl chloride (2a-p) to obtain the various silylated capsinoids
(8a-p) with different lateral chain lengths (i.e., with different degrees of
lipophilia).
Thus, the 4-t-butyldimethylsilyloxy-3-methoxybenzyl alcohol (7)
(approximately 1 mmol) was dissolved in 10-15 mL of dry pyridine in a
50 mL round-bottom flask. Argon was introduced. Next the correspond-
ing acyl chloride (2 equiv) was slowly added to this solution and was
agitated for 18 h. The reaction was monitored by TLC (eluent: 20% ethyl
acetate, 80% hexane; chromogenic reagent: anisaldehyde). The reaction
was stopped with ethyl acetate, and, after purification of the reaction
mixture, the different silylated capsinoids were obtained, as a function of
the lateral chain introduced. The products formed (silylated capsinoids
(8a-p)) and the yields are shown in Table 2.
Desilylation of Silylated Capsinoids (8a-p). The last stage (Figure 6)
was the desilylation of the various silylated capsinoids obtained pre-
viously. The t-butyldimethyl silyl group was eliminated from these
silylated capsinoids (8a-p) by the addition of a mixture of 0.25 M HCl/
ethanol (1:5). The silylated capsinoids (8a-p) were placed in a 250 mL
round-bottom flask, and approximately 80 mL of the 0.25 M HCl/ethanol
(1:5) mixture was added. Argon was introduced and the reaction mixture
was agitated for 18 h, at ambient temperature. The reaction was monitored
by TLC (eluent/20% ethyl acetate, 80% hexane; chromogenic reagent/
anisaldehyde).
Figure 2. Formation of acyl chlorides (2j,l) from their corresponding acids
(4j,l).
monitored by TLC (eluent: 20% ethyl acetate, 80% hexane; chromogenic
reagent: anisaldehyde). Then, the reaction was stopped with ethyl acetate,
and, after purification of the reaction mixture, product (6) was obtained,
which corresponds to 4-t-butyldimethylsilyloxy-3-methoxybenzaldehyde,
with a 97.56% yield (Figure 3).
Reduction of the Carbonyl of 4-t-Butyldimethylsilyloxy-3-methoxyben-
zaldehyde (6). The reaction stage (Figure 4) consists of the reduction of the
carbonyl group of 4-t-butyldimethylsilyloxy-3-methoxybenzaldehyde (6)
to its corresponding alcohol (7). With the alcohol once formed, and the
aromatic hydroxyl protected, various esterifications can be performed to
produce the capsinoids.
The reduction of the carbonyl group was performed using di-isobutyl
aluminum hydride (1 M in toluene) (DIBAL). The 4-t-butyldimethylsily-
loxy-3-methoxybenzyl (6) (3.352 g, 0.0125 mmol) was dissolved in 40 mL
of dry THF. Subsequently, 2 equiv of di-isobutyl aluminum hydride
(1 M in toluene) was slowly added, from a syringe, in an ice bath, and the
reaction mixture was stirred, under argon, at ambient temperature, for
48 h. The reaction was monitored by TLC (eluent: 20% ethyl acetate, 80%
hexane; chromogenic reagent: anisaldehyde). Then, the reaction was
stopped with water and, after purification of the reaction mixture, the pro-
duct was obtained, 4-t-butyldimethylsilyloxy-3-methoxybenzyl alcohol
(7), yield 74.42%.
Then, the reaction was stopped with, brine and, after purification of the
reaction mixture, the different capsinoids (9a-p) were obtained, as a
function of the lateral chain introduced. The capsinoids formed (9a-p)
and the reaction yields are given in Table 3.
Formation of Acyl Chlorides (2) from Their Corresponding Acids (4).
The synthesis of capsaicinoids paralleled that of the capsinoids. Several

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3345
Figure 3. Silylation of vanillin (5) (4-hydroxy-3-methoxy benzaldehyde).
Figure 4. Reduction of carbonyl of 4-t-butyldimethyl silyloxy-3-methoxy benzaldehyde (6).
Figure 5. Esterification of 4-t-butyldimethyl silyloxy-3-methoxybenzyl alcohol (7) with acyl chloride (2a-p).
Table 2. Silylated Capsinoids and Yields Obtained in the Esterification Stage

3346 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Barbero et al.
Figure 6. Desilylation of silylated capsinoids (8a-p).
solution, and it was agitated for 18 h. Then, the reaction was stopped with
ethyl acetate and, after purification of the reaction mixture, 4-t-butyldi-
methylsilyloxy-3-methoxybenzyl-(E)-8-methylnon-6-enoate (8p) was obtain-
ed, 84.15% yield.
Table 3. Synthesized Capsinoids and Yields Obtained after Desilylation
starting compound
R1
resulting compound yield (%)
8a
8b
8c
8d
8e
8f
-CH3
-CH2CH3
9a
9b
9c
9d
9e
9f
78.59
81.78
85.30
82.46
86.97
79.24
85.02
80.69
85.55
78.83
84.28
79.11
84.09
82.54
77.25
79.42
Desilylation of 4-t-Butyldimethylsilyloxy-3-methoxybenzyl-(E)-8-methyl-
6-nonenoate (8p). The final reaction was the desilylation of 4-t-butyl-
dimethylsilyloxy-3-methoxybenzyl (E)-8-methylnon-6-enoate (8p) obtained
in the preceding stage, to obtain 4-hydroxy-3-methoxybenzyl (E)-8-methyl-
non-6-enoate (9p-capsiate). The t-butyldimethyl silyl group was separated
from the 4-t-butyldimethylsilyloxy-3-methoxybenzyl (E)-8-methyl-6-none-
noate (8p), and also from the rest of the synthesized capsinoids by the
addition of a mixture of 0.25 M HCl/ethanol (1:5) (Figure 6). The 4-t-butyl-
dimethylsilyloxy-3-methoxybenzyl (E)-8-methylnon-6-enoate (8p) (0.630 g,
1.50 mmol) was placed in a 250 mL round-bottom flask, and 80 mL of
0.25 M HCl/ethanol (1:5) was added. Argon was introduced and the reac-
tion mixture was agitated for 18 h, at ambient temperature. The reaction
was monitored by TLC (eluent: 20% ethyl acetate, 80% hexane; chromo-
genic reagent/anisaldehyde).
-(CH2)2CH3
-(CH2)3CH3
-(CH2)4CH3
-(CH2)5CH3
-(CH2)6CH3
-(CH2)7CH3
-(CH2)8CH3
-(CH2)9CH3
8g
8h
8i
9g
9h
9i
8j
9j
8k
8l
-(CH2)10CH3
-(CH2)11CH3
-(CH2)12CH3
-(CH2)14CH3
-(CH2)6CH(CH3)2
9k
9l
8m
8n
8o
8p
9m
9n
9o
Then, the reaction was stopped with brine and, after purification,
4-hydroxy-3-methoxybenzyl-(E)-8-methylnon-6-enoate (9p-capsiate) was
obtained, 79.42% yield.
-(CH2)4CHdCHCH(CH3)2 9p
acids (undecanoic acid, tridecanoic acid, and 8-methyl nonanoicacid) were
transformed into their corresponding acyl chloride in order subsequently
to perform the acylations of 4-t-butyldimethylsilyloxy-3-methoxybenzyl
alcohol (Figure 2).
Synthesis of Capsiate. Capsiate (9p) is the major capsinoid found in
the “CH-19 Sweet” pepper. It has the same lateral chain as capsaicin (2),
and a double trans bond is similarly present. Hence, it is more complex to
synthesize than its preceding analogues. The synthesis has been accom-
plished using cis-8-methyl-6-nonenoic acid, which is commercial available,
as the initial reagent. Therefore, the first stage in the synthesis of capsiate
was the isomerization of the double bond to trans.
Calculation of IC50 and log P. The phytotoxicity data were fitted to a
sigmoidal dose-response model (constant slope) by employing the
GraphPad Prism v. 4.00 software package (34). c log P data were acquired
using the OSIRIS property explorer (35, 36). This software uses the Chou
and Jurs algorithm, based on computed atom contributions (37).
Bioassays. Coleoptiles were obtained from 3-day-old wheat seedlings
sown on 15 cm diameter Petri dishes fitted with Whatman No. 1 filter paper
and grown at 24 °C in the dark. The etiolated seedlings were removed from
the dishes and selected for uniformity of size under a green safelight. Then
seedlings were placed in a Van der Wij guillotine, and the apical 2 mm was
cut off and discarded. The following 4 mm lengths were selected for
bioassay and kept in an aqueous nutritive buffer for 1 h before being used,
to synchronize growth.
Products were purified (þ99%) by HPLC prior the bioassay and were
tested at 1000 μM to 10 μM in a buffered nutritive aqueous solution (citric
acid-sodium hydrogen phosphate buffer, pH 5.6; 2% sucrose). Mother
solutions of pure compounds were prepared in dimethyl sulfoxide (DMSO)
and diluted to the proper concentration with the buffer to a 0.5% v/v
DMSO final maximum concentration. The following dilutions were
prepared maintaining the same buffer and DMSO concentrations. Bio-
assays were performed in 10 mL test tubes as follows: five coleoptiles were
placed per tube each containing 2 mL of test solution; three replicates were
prepared for each test solution, and experiments were duplicated. Test
tubes were placed in a roller tube apparatus and rotated at 6 rpm for 24 h
at 22 °C in the dark. Increments of coleoptile elongation were measured
by digitalization of their photographic images, and data were statistically
analyzed.
Isomerization of cis-8-Methylnon-6-enoic Acid (4q). For the isomeriza-
tion of cis-8-methylnon-6-enoic acid (4q) a quantity of 0.310 g (1.823 mmol)
of this acid was reacted with 310 μL of 2 M NaNO2 and 215 μL of 6 M
HNO3 (Figure 7). The mixture reaction was submitted to vigorous stirring,
under argon, and was heated to 70 °C for 1.5 h. Then, the reaction mix-
ture was left to cool. Once cooled, the reaction was stopped with about
100 mL of ethyl ether, and, after purification of the mixture, a whitish oil
was obtained that corresponded to trans-8-methyl-6-nonenoic acid (4p),
98% yield.
Formation of Acyl Chloride (2p) from trans-8-Methylnon-6-enoic Acid
(4p). The procedure for the formation of the acyl chloride from trans-
8-methyl-6-nonenoic acid (4p) was the same as that described for the
capsinoids and capsaicinoids (Figure 2).
Esterification of 4-t-Butyldimethylsilyloxy-3-methoxybenzyl Alcohol
(7) with trans-8-Methylnon-6-enoyl Chloride (2p). The next reaction was
the esterification of 4-t-butyldimethylsilyloxy-3-methoxybenzyl alcohol
(7) with trans-8-methylnon-6-enoyl chloride (2p), to obtain 4-t-butyl-
dimethylsilyloxy-3-methoxybenzyl (E)-8-methylnon-6-enoate (8p). The
process is similar to that described for capsinoids (Figure 5).
RESULTS
Preparation of Capsinoids. In this study, a procedure for the
synthesis of capsinoids was developed. The starting point was
vanillin (5) and it comprises four stages: protection of the
hydroxyl group of vanillin (5) with t-butyldimethyl silyl chloride;
The 4-t-butyldimethylsilyloxy-3-methoxybenzyl alcohol (7) (0.958 g,
3.57 mmol reagent in excess) was dissolved in 15 mL of dry pyridine in a
50 mL round-bottom flask and argon was introduced. Next, trans-8-
methylnon-6-enoyl chloride (0.337 g, 1.786 mmol) was slowly added to this

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3347
Figure 7. Isomerization of cis-8-methyl-6-nonenoic acid (4q).
reduction of the carbonyl group of 4-t-butyldimethylsilyloxy-
3-methoxybenzaldehyde (6) with di-isobutyl aluminum hydride
(1 M in toluene); esterification of 4-t-butyldimethylsilyloxy-
3-methoxybenzyl alcohol (7) with acyl chlorides (2a-p); and
deprotection of the silylated capsinoids (8a-p) in order to obtain
the various capsinoids (9a-p).
The advantages of the procedure are (1) the reactions give good
yields; (2) the products obtained can be separated easily and puri-
fied; secondary reactions are not produced; (3) the reactions take
place in regioselective way; (4) the reactions are simple to put into
practice; and (5) the reagents employed are inexpensive. This makes
the procedure useful in synthesizing this family of compounds.
Characterization of Compounds. The structure of the all the
compounds synthesized was confirmed by spectroscopic methods
(1H and 13C NMR spectra; see Supporting Information). A total
of 14 capsaicinoids and 16 capsinoids were synthesized (Tables 1
and 2, respectively).
Table 4. Values of IALOGP and CLOGP for the Synthesized Capsaicinoids
and Capsinoids
capsaicinoid
IALOGP
CLOGP
capsinoid
IALOGP
CLOGP
3a
3b
3c
3d
3e
3f
0.94
1.3
-1.79
-1.16
-0.77
-0.37
0.03
0.42
0.82
1.21
1.61
2.01
2.4
9a
9b
9c
9d
9e
9f
1.26
1.71
2.18
2.66
3.17
3.7
-1.14
-0.51
-0.12
0.28
0.68
1.07
1.47
1.86
2.26
2.66
3.05
3.45
3.85
4.64
1.94
2.2
1.73
2.17
2.64
3.12
3.62
4.12
4.62
5.12
5.61
6.09
6.56
7.45
3.87
4.33
3g
3h
3i
9g
9h
9i
4.24
4.79
5.34
5.9
3j
9j
3k
3l
9k
9l
6.44
6.97
7.47
8.4
2.8
3.2
3m
3n
3p
3o
9m
9n
9p
9o
3.99
1.29
1.55
4.6
5.16
Bioassays. Bioassays were conducted on etiolated wheat co-
leoptiles for 14 capsaicinoids and 16 capsinoids, and for capsaicin
and dihydrocapsaicin. The latter two are the major capsaicinoids
present in the spicy pepper varieties and that are commercially
available.
Table 5. Chemical Characteristics of the Synthesized Capsaicinoids and
Results Obtaineda
compound IA log P IC50 ꢀ 103(mol/L) log (1/IC50) HAB HDG MW
Both the synthesized capsaicinoids and capsinoids that are
generally the most active are those that have a chain length of
8-11 carbon atoms, and a degree of lipophilia similar to that of
the natural capsaicinoids and capsinoids.
log P. Values of log P were calculated using the algorithms
IALOGP and CLOGP. IALOGP was chosen for this study,
because the commonly used algorithm CLOGP failed to give
coherent results several times. Data generated by both algorithms
are presented in Table 4.
3a
3b
3c
3d
3e
3f
0.94
1.3
2.774
1.408
0.645
0.728
0.385
0.265
0.197
0.188
0.223
1.080
0.252
0.222
0.161
0.625
0.714
4.062
2.5569
2.8514
3.1902
3.1379
3.4146
3.5770
3.7051
3.7251
3.6525
2.9666
3.5979
3.6527
3.7937
3.2041
3.1460
2.3912
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
195
209
223
237
251
265
279
305
293
307
307
321
335
349
363
391
1.73
2.17
2.64
3.12
3.62
3.87
4.12
4.33
4.62
5.12
5.61
6.09
6.56
7.45
3g
3p
3h
3o
3i
DISCUSSION
A series of 14 analogues of capsaicinoids with different linear
chain lengths (2a-p) were synthesized (Table 1), by amidation of
4-hydroxy-3-methoxy benzylamine hydrochloride with the acyl
chlorides of these linear acids. Similarly, 16 analogues of capsinoids
(9a-p) have been synthesized by esterification of vanillyl alcohol.
The degree of lipophilia of the synthesized capsaicinoids in-
creased linearly with the increased chain length. Therefore, a series
of capsaicinoids and capsinoids have been obtained that present a
wide range of values of log P. The log P values of the natural
capsaicinoids (capsaicin and dihydrocapsaicin) and those of the
natural capsinoids (capsiate and dihydrocapsiate) lie in intermedi-
ate positions among the values of all the compounds synthesized.
The IC50 values for the capsaicinoids assayed were calculated,
using a sigmoidal dose-response model of constant slope, from
the activities obtained in the wheat coleoptile assay. These IC50
values are expressed in mol/L. The chemical characteristics of the
synthetic capsaicinoids and their effects are presented in Table 5.
Table 6 shows the results for the caspsinoids.
3j
3k
3l
3m
3n
a HAB (H acceptor bonds); HDG (H donors groups); MW (molecular weight).
noted that the products 3o and 3k were not well fitted to eq 1;
consequently, they were discarded for the calculation of this
equation. To obtain eq 2, the compounds 9p and 9m were
discarded, for the same reason. In both cases, the correlation
coefficients are >0.95, at 0.9696 and 0.9521 for eqs 1 and 2,
respectively, for 14 samples (n = 14).
2
logð1=IC50Þ ¼ -0:1108ðIAlogPÞ þ 0:9109ðIAlogPÞ þ 1:8213
ð1Þ
2
In this study, the IA log P values and the IC50 values were
correlated and expressed in the form [log(1/IC50)] for all the
compounds prepared. A linear fit, logarithmic, and second-order
polynomial, was performed. The values obtained show a very
good fit to a second-order polynomial, fitted using a second-order
equation (eqs 1 and 2). In the case of the capsaicinoids, it was
logð1=IC50Þ ¼ -0:0881ðIAlogPÞ þ 0:8919ðIAlogPÞ þ 1:3394
ð2Þ
In light of Figure 8, it is noted that the capsaicinoid presenting
the most activity is capsaicin (3p); this is the major natural
capsaicinoid. Capsaicinoids 3g and 3h also present excellent

3348 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Barbero et al.
Table 6. Chemical Characteristics of the Synthesized Capsinoids and Results
Obtaineda
compound IA log P IC50 ꢀ 103(mol/L) log(1/IC50) HAB HDG MW
9a
9b
9c
9d
9e
9f
1.26
1.71
2.18
2.66
3.17
3.7
3.638
2.835
1.170
1.210
0.601
0.386
0.356
0.517
0.186
0.220
0.181
0.309
0.467
0.559
2.433
2.451
2.4391
2.5474
2.9318
2.9172
3.2210
3.4132
3.4487
3.2863
3.7305
3.6570
3.7423
3.5100
3.3305
3.2527
2.6138
2.61066
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
196
210
224
238
252
266
280
306
294
308
308
322
336
350
364
392
9g
9p
9h
9o
9i
4.24
4.6
4.79
5.16
5.34
5.9
9j
9k
9l
6.44
6.97
7.47
8.4
9m
9n
Figure 9. Representation of IAlog P vs log(1/IC50) for the synthesized
capsinoids.
a HAB (H acceptor bonds); HDG (H donors groups); MW (molecular weight).
With respect to the biological properties of the capsaicinoids
and capsinoids, it was observed (Figures 8 and 9) that the two
families of compounds exhibit similar biological properties. Both
similar inhibit coleoptile growth, in those products with the most
activity. The only observable difference between capsaicinoids
and capsinoids is the following: in the capsaicinoids, the maxi-
mum activity is with products having 8 and 9 carbon atoms in the
lateral chain (3.62<IAlog P<4.12), including capsaicin, but, in
the capsinoids, the maximum activity is with those having 9 and
10 carbon atoms in the lateral chain (4.79 < IAlog P < 5.34).
The synthesis of natural capsaicinoids and capsinoids can also
be performed chemically, but the nature of their lateral chains
(methyl terminals and double links) makes their synthesis com-
plicated and very costly. The possibility of obtaining these con-
geners much more easily, and economically, means that they may
be of commercial value in the food and pharmaceutical industries.
Supporting Information Available: Details of capsaicinoids
and capsinoids. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 8. Representation of IAlog P vs log(1/IC50) for synthesized caps-
aicinoids.
LITERATURE CITED
activity; these products have lipophilia values very similar to that of
capsaicin. A similar effect was observed in Figure 9, where the most
active products are 9h, 9i, and the natural capsinoid, dihydrocapsiate
(9o). In general, the further the lipophilia values (log P) were from
those found naturally, whether higher or lower, the more reduced
were the biological properties of the synthesized compounds.
The results show that, out of all the capsaicinoids and capsi-
noids assayed, the natural capsaicinoids and capsinoids, and
those synthetic compounds with lipophilia values similar to those
of the natural products, are the most active biologically. This is
more important in the case of capsinoids; although these natural
compounds are found in peppers, very few varieties of pepper
possess congeners. To obtain sufficient volume of these capsi-
noids to take advantage of their beneficial properties, they will
need to be synthesized.
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pounds, instead of linear, more accessible fatty acids. A similar
case exists in sorgoleone, whose analogues with simpler chains
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been suggested that the plant possibly utilizes these transforma-
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