A new approach for the total synthesis of spilanthol and analogue with improved anesthetic activity

Article abstract of DOI:10.1016/j.tet.2018.06.034

A 5-step synthesis of spilanthol (affinin) is reported, where the route features complete control of alkene geometry during the assembling of the double bonds, with the use of a Sonogashira cross-coupling reaction, a Z-selective alkyne semi-reduction and a HWE olefination reaction as the key steps. A simplified analogue was also prepared in 4 steps. Both compounds were found to permeate dermatomed pig ear skin through an in vitro Franz-type diffusion cell. The simplified analogue presented a superior anesthetic effect in vivo, using the tail flick model, when compared to spilanthol and to the commercial standard EMLA. These results suggest that both spilanthol and its analogue could be useful as a topical anesthetic in clinical practice.

Full text of DOI:10.1016/j.tet.2018.06.034

Accepted Manuscript
A new approach for the total synthesis of spilanthol and analogue with improved
anesthetic activity
Isabella G. Alonso, Lais T. Yamane, Verônica S. de Freitas-Blanco, Luiz F.T.
Novaes, Michelle F.M.B. Leite, Eneida de Paula, Marili V.N. Rodrigues, Rodney A.F.
Rodrigues, Julio C. Pastre
PII:
S0040-4020(18)30733-6
10.1016/j.tet.2018.06.034
TET 29630
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 20 February 2018
Revised Date: 12 June 2018
Accepted Date: 14 June 2018
Please cite this article as: Alonso IG, Yamane LT, de Freitas-Blanco VerôS, Novaes LFT, Leite MFMB,
de Paula E, Rodrigues MVN, Rodrigues RAF, Pastre JC, A new approach for the total synthesis
of spilanthol and analogue with improved anesthetic activity, Tetrahedron (2018), doi: 10.1016/
j.tet.2018.06.034.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A new approach for the total synthesis of
spilanthol and analogue with improved
anesthetic activity.
Leave this area blank for abstract info.
Isabella G. Alonsoa, Lais T. Yamaneb, Verônica S. de Freitas-Blancob, Luiz F. T. Novaesa, Michelle F. M. B.
Leitec, Eneida de Paulad, Marili V. N. Rodriguesb, Rodney A. F. Rodriguesb and Julio C. Pastrea,
aInstitute of Chemistry, University of Campinas, P.O. Box 6154, Campinas, SP 13084-971, Brazil
bChemical Biological and Agricultural Research Center (CPQBA), University of Campinas, Campinas, SP
13148-218, Brazil
cInstitute of Biology, University of Campinas, Campinas, SP 13083-862, Brazil
dSchool of Dentistry at Piracicaba, University of Campinas, Piracicaba, SP 13414-903, Brazil

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
A new approach for the total synthesis of spilanthol and analogue with improved
anesthetic activity
Isabella G. Alonsoa, Lais T. Yamaneb, Verônica S. de Freitas-Blancob, Luiz F. T. Novaesa, Michelle F. M.
B. Leitec, Eneida de Paulad, Marili V. N. Rodriguesb, Rodney A. F. Rodriguesb and Julio C. Pastrea,
aInstitute of Chemistry, University of Campinas, P.O. Box 6154, Campinas, SP 13084-971, Brazil
bChemical Biological and Agricultural Research Center (CPQBA), University of Campinas, Campinas, SP 13148-218, Brazil
cInstitute of Biology, University of Campinas, Campinas, SP 13083-862, Brazil
dSchool of Dentistry at Piracicaba, University of Campinas, Piracicaba, SP 13414-903, Brazil
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A 5-step synthesis of spilanthol (affinin) is reported, where the route features complete control
of alkene geometry during the assembling of the double bonds, with the use of a Sonogashira
cross-coupling reaction, a Z-selective alkyne semi-reduction and a HWE olefination reaction as
the key steps. A simplified analogue was also prepared in 4 steps. Both compounds were found
to permeate dermatomed pig ear skin through an in vitro Franz-type diffusion cell. The
simplified analogue presented a superior anesthetic effect in vivo, using the tail flick model,
when compared to spilanthol and to the commercial standard EMLA®. These results suggest that
both spilanthol and its analogue could be useful as a topical anesthetic in clinical practice.
Available online
Keywords:
Anesthetic
Tail flick
Spilanthol
2009 Elsevier Ltd. All rights reserved
.
Total synthesis
Sonogashira reaction
———
Corresponding author. Tel.: +55 19 35213143; e-mail: juliopastre@iqm.unicamp.br

2
Tetrahedron
1. Introduction
present a great variety of compounds with fluctuating contents
ACCEPTED MANUSCRIPT
thereof, depending on the extraction procedure or seasonal
factors, we envisioned further exploring the anesthetic activity
with spilanthol obtained by chemical synthesis.
Novel chemical structures can be isolated from natural
sources, and they have been applied for drug discovery over the
past decades with great success. Indeed, between 1981 and 2014,
natural products and molecules inspired by their scaffolds
represent more than half of all approved small-molecule drugs.1
Herein, we report a new synthetic approach to obtain
spilanthol in high purity, as well as a simplified analogue. The
permeation profiles of both compounds were evaluated through
in vitro experiments with a Franz-type diffusion cell, and their
anesthetic effect was assayed in vivo using the tail flick model.
Spilanthol, also known as affinin (Figure 1), is a member of
the N-alkylamide family and has been found in several natural
sources, such as Acmella oleracea (jambu), A. ciliate, A.
oppositifolia, A. radicans, Welelia parviceps and Heliopsis
longipes.2,3 Spilanthol is a high-value added compound and is
commercially available, however, it is sold with approximately
80% isomeric purity at prices as high as $150.00 for 1 mg or
$1,400.00 for 10 mg.4
2. Results and discussion
2.1. Chemical synthesis
Since its first isolation by Gerber in 1903,27 four total
syntheses of spilanthol have been reported to date, and these
works are summarized in Figure 2A. The first successful
synthesis was reported by Crombie and co-workers in 1963,28
allowing for the structural elucidation regarding the double bonds
stereochemistry for this natural product.
Extracts containing spilanthol, or even the aerial parts of
jambu, have been used for centuries in traditional folk medicine
and cuisine throughout the world.5 Thus, several studies
performed with spilanthol, or most commonly with enriched
plant extracts, exhibited an array of biological activities,
including antipyretic,6 diuretic,7,8 antimalarial,9 aphrodisiac,10,11
anti-inflammatory12,13 and interestingly, analgesic and anesthetic
activity, among many others.5,13-14
Yamamoto and co-workers approached the synthesis using a
convergent strategy with the addition of allyltitanium species
over an aldehyde as a key step. Spilanthol was obtained in 88%
purity determined by GC-FID analysis.29,30
Local anesthetics, one of the most important advancements in
medicine, are chemical substances that cause a reversible loss of
sensation due to a blockade of nerve conduction around the site
of application.16 When topically applied, anesthetics produce a
superficial loss of sensation in the conjunctiva, mucous
membranes or skin and, therefore, are used in numerous medical
In 1998, Lu and co-workers reported the shortest synthesis of
spilanthol, where the key transformation employed was a
cotrimerization of acrolein with two equivalents of acetylene,
which assembled the conjugated diene fragment with the required
geometry in 43% yield. However, the use of acetylene is a
drawback of this synthesis, since this gas is extremely flammable
and potentially explosive, posing some safety concerns on scale.
specialties,
such
as
dentistry,
otorhinolaryngology,
ophthalmology, urology, aesthetic surgery, among others, to
minimize the anxiety, pain or discomfort caused by some
procedures.16,17
From 2009 to 2012, Takasago International Corporation has
filed several patents for the synthesis and use of spilanthol.31-35
The synthesis was developed on multi-gram scale, producing
67.0 g of spilanthol from a single batch, and only extraction,
distillation and crystallization were required for the purification
steps. However, a poor control over the double bond formation
was observed: spilanthol was obtained with 78% for the desired
isomer, 18.0% for the all-E isomer, and 4% for the (2E,6Z,8Z)-
isomer.
The most widely used topical anesthetics are lidocaine,
benzocaine and prilocaine in form of gels, creams, sprays,
solutions or ointments. EMLA® (Eutectic Mixture of Local
Anesthetics), which is composed of 2.5% of lidocaine and 2.5%
of prilocaine (Figure 1), is often regarded as the gold standard
for topical anesthesia.18
Bearing these syntheses and their limitations in mind, we
sought to develop a short synthesis of spilanthol and some simple
analogues which would not require the use of extremely toxic or
dangerous chemicals, and could provide spilanthol with good
control of alkene geometry. Thus, we proposed the retrosynthesis
depicted in Figure 2B. Initially, the C2-C3 double bond would
be forged by a Horner-Wadsworth-Emmons (HWE) olefination.
The product of this reaction is usually easier to purify when
compared to a Wittig reaction, the choice of Yamamoto and Lu,
which produces triphenylphosphine oxide and can require
multiple purifications.36
Fig. 1. Chemical structures of lidocaine, prilocaine and spilanthol.
Despite being generally regarded as safe, the use of topical
anesthetics requires caution. When the recommended dose is
exceeded, systemic effects may occur, with the patient presenting
symptoms such as dizziness, respiratory distress, loss of
consciousness and even cardiac arrest.19 Moreover, there are
reports of allergic reactions, neurotoxicity, and cardiotoxicity that
led some patients to death.20-22
The C6-C7 olefin could arise from a selective semi-reduction
of alkyne 2, which in turn could be generated via a Sonogashira
cross-coupling reaction between alkyne 5 and 1-bromo-1-
propene as a mixture of isomers (E/Z 60:40), which is nearly 20
times cheaper than the (E)-1-bromo-1-propene. The production
of the desired E-isomer of compound 2 relied on previous studies
which demonstrated that (E)-1-bromo-1-propene reacts faster in
Sonogashira reactions than the Z-isomer,37 and that double bond
isomerization can occur under certain reaction conditions.38,39
In this scenario, the investigation of safer topical anesthetics is
highly desired and timely. As far as spilanthol is concerned, it is
considered safe by the European Food Safety Authority (EFSA)
and jambu oleoresin is classified as a Generally Recognized as
Safe (GRAS) substance by the Flavor and Extract Manufacturers
Association (FEMA), being included in several alimentary
products.23-24
Synthesis of fragment 3 was planned to be conducted with a
direct amidation of the corresponding ethyl ester with
isobutylamine.
In our previous work, we found that ethanolic extracts of
aerial parts of Acmella oleracea exhibited significant anesthetic
activity, similar to the EMLA® effect.25,26 Since extracts usually

3
Analogue 8 would be prepared via a similar route, except for
the absence of alkyne semi-reduction (Figure 2C). The alkyne
was thought to give more rigidity to the molecule, which could
impact its bioactivity and stability profile.
Morimoto, Ohshima and co-workers could be beneficial to
ACCEPTED MANUSCRIPT
synthesize this phosphonoacetamide.45 The reaction was carried
out in the absence of solvent, with a small excess of
isobutylamine, in the presence of catalytic amounts of
lanthanum(III) triflate at room temperature. Phosphonoacetamide
3 was obtained in 93% yield and no side-products were observed,
which facilitated its purification.
Next, we turned our attention to the conjugated diene
fragment (Scheme 1B). The carbon skeleton was constructed
using a Sonogashira cross-coupling reaction between 4-pentyn-1-
ol and 1-bromo-1-propene as a mixture (E/Z 60:40).46
Our first set of optimizations for the Sonogashira reaction
used vinyl bromide 4 as the limiting reactant and excess of
alkyne 5. In an attempt to favor double bond isomerization,
experiments performed at higher temperatures (i.e. 100 °C)
provided low selectivities and yields (Table 1, entries 2-4).
When the reaction was performed at 60 °C (Table 1, entries 5-9)
higher selectivities were obtained, although the yields were low
for most of the bases investigated. Curiously, the use of
piperidine as base furnished the product in almost quantitative
yield (entry 9), but with marginal isomerization favoring the
undesired isomer, while the use of diisopropylamine (DIPA,
entry 5) led to the highest selectivity, with a moderate yield. As
vinyl bromide 4 is a volatile compound (b.p. 58-63 °C), we also
attempted the reaction at room temperature (entry 1), which
provided a similar result to the test at 60 °C. Due to easier
experimental setup, it was chosen as the best condition at this
point and the reaction was further investigated (Table 2).
Entry a
Deviation from the above
None
E:Z Ratio b
91:9
Yield (%) c
1
2
3
4
5
6
7
8
9
56
44
77
64
45
64
55
39
98
100 °C
81:19
42:58
77:23
93:7
100 °C, K2CO3 as base
100 °C, Et3N as base
60 °C
60 °C, Cs2CO3 as base
60 °C, K2CO3 as base
60 °C, Et3N as base
60 °C, Piperidine as base
60:40
57:43
88:12
54:46
Table 1. Optimization of the key Sonogashira reaction.
a Scale: 1 mmol of 1-bromo-1-propene (E/Z 60:40), using a sealed tube, up to
b
1
c
15 h. Determined by H NMR analysis of the crude. Isolated yield for
duplicates.
For the second round of optimizations, alkyne 5 was
employed as the limiting reactant without significant change in
yield and selectivity (Table 2, entry 1). Triphenylphosphine was
found as an important additive to further increase the selectivity
towards the E-alkene 2; this reagent decreased the formation of
dark solid, which was attributed to palladium black, and the next
modifications included the presence of PPh3. Also, we observed
an increase in the reaction kinetics as the reaction went to
completion in just 2 hours.
From the evaluated solvents, DMF favored
a higher
selectivity, ranging from 94:6 to >95:5 with moderate yield
(entries 2-4). Use of Pd(PPh3)2Cl2 instead of Pd(PPh3)4 or
decreasing the palladium catalyst loading led to smaller
selectivities or yields (entries 5-7). Notably, entry 3 presents
higher yield, but was not chosen during the spilanthol synthesis
because the minor isomer could not be separated by standard
techniques, as pointed out previously. Although the yield could
not be improved beyond 53%, DIPA was the base of choice since
it furnished the best compromise between yield and
diastereoselectivity of the product. Under these best conditions,
the desired 6E-isomer of 2 was obtained in 53% yield in more
than 95:5 diastereomeric ratio, as determined by 1H NMR
analysis.
Fig. 2. (A) Previous syntheses of spilanthol; (B) retrosynthesis of spilanthol
and (C) analogue 8 in this work.
Initially, we explored the synthesis of (2-(isobutylamino)-2-
oxoethyl)phosphonate fragment 3 (Scheme 1A). This scaffold
has been synthesized by different methods which required
multiple steps, the use of toxic reagents or furnished moderate
yields.40-44 We envisioned that a methodology described by

4
Tetrahedron
ACCEPTED MANUSCRIPT
Table 2. Further optimization of the key Sonogashira
reactiona
a Scale: 1 mmol of 4-pentyn-1-ol unless noted otherwise. b Determined by 1H
NMR analysis of the crude. c Isolated yield for duplicates. d Scale: 1 mmol to
10 mmol of 4-pentyn-1-ol; lower selectivities were obtained on larger scales.
e 15 h.
Entry a
Deviation from the above
No PPh3
E:Z Ratio b
91:9
Yield (%) c
1
2d
3
56
53
77
38
49
20
11
None
93:7 to > 95:5
79:21
The diastereoselectivity obtained for the Sonogashira cross-
coupling reaction was confirmed by the use of pure (E)-1-bromo-
1-propene (obtained after isomerization of the E/Z mixture under
basic conditions)39 and (Z)-1-bromo-1-propene (obtained from
commercial sources). Thus, employing piperidine as base in
DMF at room temperature, the Sonogashira adducts (E)- and (Z)-
2 were obtained in 81% and 68%, respectively, without any
noticeable double bond isomerization (>95:5 d.r. according to 1H
THF as solvent
4
MeCN as solvent
Pd(PPh3)2Cl2 as Pd source
2.5 mol% of Pd(PPh3)4
1.0 mol% of Pd(PPh3)4
84:16
5
83:17
6e
7e
>95:5
78:22
1
NMR analysis). The peaks at 6.04 and 5.90 ppm in the H NMR
spectra were unequivocally attributed to the (E)- and (Z)-isomers,
respectively, and allowed an easy diastereomeric ratio assessment
(see Figures S7, S10, S12 and S14 in the Supporting
Information).
The subsequent step to produce the key diene 9 involved a Z-
selective alkyne semi-reduction with an alloy of zinc, copper and
silver in the presence of trimethylsilyl chloride (modified
Boland’s protocol), based on the report of Hansen.47 The alkyne
reduction proceeded in 59% yield with complete selectivity
towards the expected Z-isomer (>95:5 by 1H NMR analysis).
Finally, the conclusion of the spilanthol synthesis required the
connection of the fragments and generation of a third double
bond with E-geometry. To accomplish this task, alcohol 9 was
subjected to a Swern oxidation, and the crude aldehyde was then
employed in
a Horner-Wadsworth-Emmons reaction with
phosphonoacetamide 3. The newly created alkene exclusively
1
presented an E-geometry (> 95:5 by H NMR analysis) and
spilanthol was thus obtained in 63% yield, over two steps. Its
spectroscopic data (1H and 13C NMR) were identical to those of
an authentic sample of spilanthol obtained from a commercial
source (see Supporting Information for details).
The synthesis of analogue 8 required a similar sequence
(Scheme 1C), but no alkyne reduction was necessary. For this
final product, the minor isomer constituted by the alkene with
8Z-geometry could be removed after standard flash
chromatography. On the other hand, the minor 8Z-isomer of
spilanthol could not be removed by the same procedure, and for
this reason, we extensively studied the initial Sonogashira
coupling aiming for a higher E-selectivity.
Scheme 1. Synthesis of spilanthol and analogue 8
2.2. Biological evaluation
2.2.1. In vitro permeation studies
Aiming for the application of spilanthol and analogue 8 as
topical anesthetics, both compounds were incorporated in the
formulation of bioadhesive films (see experimental section for
details). The in vitro permeability was evaluated using Franz-
type vertical diffusion cells, an apparatus commonly used to
evaluate the permeation of compounds through the skin or
mucosa.48 As a barrier, dermatomed pig ear skin was used, due to
the similarities between the pig tissues and the human skin.49
Other research groups have described that spilanthol was able
to permeate the skin and mucosa, and could also act as an
enhancer, increasing the permeation of some substances through
the dermis.50-52 In our study, spilanthol and analogue 8 showed a
similar permeation profile through dermatomed pig ear skin, as
can be seen in Figure 3 and the permeation profile of spilanthol
was compatible with those observed in the literature.51 The flux
(spilanthol: 0.78 ± 0.11, analogue 8: 0.88 ± 0.09 ꢀg.cm−2.h−1) and

5
lag time (spilanthol: 0.66 ± 0.41, analogue 8: 0.81 ± 0.43 h) for
spilanthol and analogue 8 were identical within the limits of
experimental error.
a short onset of action (5 min). Both compounds demonstrated
ACCEPTED MANUSCRIPT
anesthesia similar or superior to EMLA®, the anesthetic currently
considered as the gold standard in topical anesthesia. It is
important to highlight that the effect observed for spilanthol and
its analogue required extremely low doses (approximately 100
ꢀg).
A)
B)
Fig. 3. Permeation profiles of spilanthol and analogue 8 applied under finite
dose conditions (mean values ± SEM)
This result can be understood from the similar molecular
structures and physical parameters, such as log P and polar
surface area which both compounds share. Since spilanthol and
analogue 8 exhibited similar permeation profiles, we then
evaluated their anesthetic activity.
2.2.2. In vivo anesthetic effect
The tail flick model was employed for the anesthetic
evaluation of spilanthol and analogue 8. This model, first
described by D’Amour and Smith,53 has been used to evaluate the
anesthetic effect of topical formulations by several groups.54-56
Fig. 4. Effects of topical administration of EMLA®, spilanthol and analogue 8
in the tail flick assay. Time-course (min) showing the percent of animals with
anesthesia (A) and the duration of anesthesia (B). (n = 5-6/group). Data
expressed as mean±SEM AUC for MPE (%) p< 0.001 when spilanthol and
EMLA were compared to analogue 8. Duration of anesthesia: **p<0.01. One-
way analysis of variance with Tukey posthoc test.
The bioadhesive films containing spilanthol and analogue 8
were prepared according to our previous work using the natural
polymer hydroxyethyl cellulose (HEC) and some adjuvants57 (see
experimental section and Supporting Information for more
details). Initially, a bioadhesive film composed of hydroxyethyl
cellulose, Tween® 80, glycerin and Transcutol®, prepared without
an anesthetic compound was evaluated as a negative control, and
showed no antinociceptive effect (data not shown in Figure 4),
which confirmed that the film basis is inactive.
3. Conclusions
This work combined modern synthetic chemistry and
bioassays to address the development of anesthetics for dentistry,
surgeries, among other applications. We presented a new route
for the synthesis of the N-alkylamide spilanthol, which features
complete control over the geometry of its three double bonds. A
simplified analogue was also prepared with an alkyne replacing
the Z-alkene, which required one step less when compared to
spilanthol. Both compounds presented similar profiles for in vitro
permeation across dermatomed pig ear skin. Their anesthetic
profile was evaluated in vivo with the tail flick model, and
analogue 8 showed a superior effect when compared to spilanthol
and the commercial anesthetic EMLA®. These results open the
door to exploration of new spilanthol derivatives and further
investigations of compound 8 as a potential tool for medicine.
We observed through the tail flick test that spilanthol and
EMLA® showed similar anesthesia duration (Figure 4), while
analogue 8 presented an increased anesthetic effect (p<0.01).
Taking into account the previous studies with jambu extracts, our
work confirms that the antinociceptive activity is indeed due to
the presence of spilanthol, and not to an association of several
chemical components.
In view of the superior activity exhibited by analogue 8, we
hypothesized that the alkyne group in compound 8 imposes
conformational constraints on the molecule when compared to
spilanthol, which might enhance affinity or selectivity with a pain
receptor. Further studies are being conducted by our group in
order to elucidate the mode of action of spilanthol and analogues.
4. Experimental section
4.1. Chemical synthesis
Despite the recent advances in topical anesthesia, where a
variety of new drug delivery systems have been developed to
enhance and improve anesthesia, topical anesthetics remain
underused.58 This scenario is partly due to the long onset of
action that most topical anesthetics require, and also due to a lack
of effectiveness in some procedures.59 The results presented here
showed that analogue 8 and spilanthol are potent anesthetics with
Starting materials and reagents were obtained from
commercial sources and used as received unless otherwise
specified. Dichloromethane, triethylamine, diisopropylamine
(DIPA) were treated with calcium hydride and distilled before
use. Tetrahydrofuran (THF) was treated with metallic sodium

6
Tetrahedron
and benzophenone and distilled before use. Dry N,N-
6.2 Hz, 2H), 5.43 (dsext, J = 15.8, 1.9 Hz, 1H), 6.04 (dq, J =
ACCEPTED MANUSCRIPT
dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were
obtained from Aldrich. Anhydrous reactions were carried out
with continuous stirring under an atmosphere of dry nitrogen.
Progress of the reactions was monitored by thin-layer
chromatography (TLC) analysis (Merck, silica gel 60 F254 on
aluminum plates), unless otherwise stated. Flash chromatography
purifications were performed with silica gel 60, 220-440 mesh,
15.8, 6.8 Hz, 1H). 13C NMR (62.9 MHz, CDCl3): δ 15.8 (CH2),
18.3 (CH3), 31.3 (CH2), 61.7 (CH2), 79.7 (C), 87.4 (C), 110.8
(CH), 138.3 (CH). Data obtained for the (Z)-isomer: H NMR
1
(CDCl3, 400 MHz): δ 1.57 (s, 1H), 1.81 (quint, J = 6.5 Hz, 2H),
1.85 (dd, J = 6.8, 1.7 Hz, 3H), 2.49 (td, J = 6.9, 2.1 Hz, 2H), 3.79
(t, J = 6.1 Hz, 2H), 5.45 (d sext, J = 10.7, 1.9 Hz, 1H), 5.90 (dq, J
= 9.7, 6.8 Hz, 1H). 13C NMR (101 MHz, CDCl3): δ 15.7 (CH3),
16.1 (CH2), 31.5 (CH2), 61.9 (CH2), 77.8 (C), 93.8 (C), 110.1
(CH), 137.3 (CH).
Sigma-Aldrich. H NMR, 13C NMR and 31P NMR spectra were
1
recorded on Bruker 250, 400 and 600 MHz spectrometers, the
chemical shifts (δ) were reported in parts per million (ppm)
relative to deuterated solvent as the internal standard (CDCl3:
4.1.3. (4Z,6E)-octa-4,6-dien-1-ol (9)
7.26 ppm for H NMR and 77.0 ppm for 13C NMR), coupling
1
Zinc dust (5.00 g, 76 mmol, 76 eq.) and H2O (30 mL) were
added to a round-bottomed flask, and argon was bubbled through
the mixture with stirring at room temperature for 15 min. Next,
the bubbling was stopped and the argon atmosphere was
maintained, Cu(OAc)2•H2O (1.00 g, 4.9 mmol, 4.9 eq.) was
added by removing the septum and adding the solid quickly and
the reaction mixture allowed to stir for another 15 min. Then
AgNO3 (600 mg, 3.5 mmol, 3.5 eq.) was added by removing the
septum and adding the solid quickly. This step is exothermic and
temperature of the reaction mixture was slightly increased. The
reaction was stirred for 30 min. Then the solid was separated by
filtration under a stream of nitrogen, and was washed with H2O
(3 x 25 mL), methanol (3 x 25 mL), acetone (3 x 25 mL), and
Et2O (3 x 25 mL). The alloy still moist with Et2O was collected
and added to a solution of alkyne 2 (124 mg, 1.0 mmol, 1 eq.)
diluted in MeOH/H2O (15 mL, 1:1) under a N2 atmosphere, and
TMSCl (1.3 mL, 10 mmol, 10 eq.) was added. The reaction was
stirred for 30 h and a second portion of TMSCl (1.3 mL, 10
mmol, 10 eq.) was added. The reaction was stirred for a second
period of 30 h, then the reaction mixture was filtered through a
column of Celite and the solid was washed with EtOAc (4 x 80
mL). The liquid phase was washed with brine (50 mL), the
organic phase was dried (MgSO4), concentrated in vacuo and the
crude product was purified by flash chromatography (SiO2,
hexanes:EtOAc, 75:25) to furnish the diene 9 (74.4 mg, 0.59
mmol) as a colorless oil in 59% yield. Rf 0.46 (SiO2,
constants (J) are in hertz (Hz). The following abbreviations were
used to designate chemical shift multiplicities: s = singlet, d =
doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br.
= broad signal. NMR spectra were processed using ACD/NMR
Processor Academic Edition version 12.01. High resolution mass
spectra (HRMS) were recorded on a Waters Xevo Q-Tof
apparatus operating in positive ion electrospray mode (ESI+).
Fourier Transform Infrared (FTIR) spectra were recorded on a
Thermo Scientific Nicolet iS5, the principal absorptions are listed
in cm−1. IUPAC names of the compounds were generated using
ChemBioDraw Ultra 12.0.
4.1.1. Diethyl (2-(isobutylamino)-2-
oxoethyl)phosphonate (3)
A 5 mL round-bottomed flask with a magnetic stirrer was
charged with lanthanum (III) triflate hydrate (La(OTf)3•xH2O,
325 mg, 10 mol%), the flask was heated with a heat-gun over 220
°C for 15 min under vacuum (<1 mbar), and was filled with dry
N2. Next, triethyl phosphonoacetate (7, 1.136 g, 4.9 mmol, 1.0
eq.) and isobutylamine (6, 507 mg, 6.9 mmol, 1.4 eq.) were
added via syringe (no exothermic reaction was observed during
addition of all chemicals). The reaction was stirred for 4 h at
room temperature, then the crude mixture was filtered through a
column of silica using CH2Cl2:MeOH (100:0 to 90:10) as eluent
to furnish the amide 3 (1.15 g, 4.6 mmol) as a colorless oil in
93% yield. Rf 0.19 (SiO2, EtOAc). 1H NMR (250 MHz, CDCl3):
δ 0.85 (d, J = 6.6 Hz, 6H), 1.27 (t, J = 7.1 Hz, 6H), 1.72 (non, J =
6.9 Hz, 1H), 2.75 (s, 1H), 2.83 (s, 1H), 3.02 (t, J = 6.3 Hz, 2H),
4.07 (dq, J = 8.2, 7.1 Hz, 4H), 6.92 (s, 1H). 31P NMR (101 MHz,
CDCl3): δ 22.8-23.5 (m, 1P). 13C NMR (62.9 MHz, CDCl3) δ:
16.2 (d, JC-P = 6.4 Hz, CH3), 19.9 (2xCH3), 28.2 (CH), 34.8 (d,
JC-P = 131 Hz, CH2), 47.0 (CH2), 62.5 (d, JC-P = 6.9 Hz, 2CH2),
165.8 (d, JC-P = 3.2 Hz, C).
1
hexanes/EtOAc 60:40). H NMR (250 MHz, CDCl3): δ 1.51 (br.
s, 1H), 1.66 (quint., J = 7.0 Hz, 2H), 1.77 (d, J = 6.5 Hz, 3H),
2.25 (q, J = 7.2 Hz, 2H), 3.66 (t, J = 6.5 Hz, 2H), 5.29 (dt, J =
10.7, 7.6 Hz, 1H), 5.68 (dq, J = 14.9, 6.6 Hz, 1H), 5.97 (t, J =
10.9 Hz, 1H), 6.04 (ddt, J = 15.0, 11.1, 1.3 Hz). 13C NMR (62.9
MHz, CDCl3): δ 18.7 (CH3), 23.9 (CH2), 32.4 (CH2), 62.2 (CH2),
126.7 (CH), 128.5 (CH), 129.1 (CH), 129.5 (CH).
4.1.4. Spilanthol (1)
4.1.2. (E)-oct-6-en-4-yn-1-ol (2)
A round-bottomed flask under a N2 atmosphere was charged
with dry CH2Cl2 (8 mL) and oxalyl chloride (144 ꢀL, 1.7 mmol,
2 eq.). The solution was cooled to −78 °C, and anhydrous DMSO
(181 ꢀL, 2.5 mmol, 3 eq.) was added (evolution of gas was
observed). The reaction was stirred for 15 min, then alcohol 9
(107 mg, 0.85 mmol, 1 eq.) was added to the mixture as a
solution in CH2Cl2 (2 mL + 2 mL of washing). The resulting
solution was stirred for 1 h at −78 °C. Next, dry Et3N (711 ꢀL,
4.2 mmol, 6 eq.) was added, the cooling bath was removed and
the reaction was further stirred for 30 min. The reaction was
diluted with Et2O (15 mL), and the organic phase was extracted
with H2O (2 x 10 mL), brine (10 mL), dried (MgSO4), and
concentrated under reduced pressure (100 mbar, 25 °C, due the
volatility of aldehyde). The crude product was used in the HWE
step without further purification.
A round-bottomed flask was charged with PPh3 (347 mg, 1.3
mmol, 10 mol%), CuI (382 mg, 2.0 mmol, 15 mol%) and
Pd(PPh3)4 (757 mg, 0.65 mmol, 5 mol%), the atmosphere was
exchanged for dry N2. Next, anhydrous DMF (26 mL),
diisopropylamine (3.7 mL, 26 mmol, 2 eq.), 1-bromo-1-propene
(4, 60:40 mixture of E/Z isomers, 1.4 mL, 16 mmol, 1.2 eq.) and
pent-4-yn-1-ol (5, 1.3 mL, 13 mmol, 1 eq.) were added to the
flask in this order, at room temperature. The reaction was stirred
for 24 h at room temperature, then H2O (200 mL) and Et2O (200
mL) were added, the mixture was shaken vigorously, the organic
phase was separated, washed with H2O (100 mL) and brine (100
mL), dried (MgSO4) and concentrated in vacuo (35 °C, 600-50
mbar). A sample was taken to measure the E/Z ratio of the
product (ranging from 93:7 to >95:5 of E/Z ratio). Next, the
crude was purified by flash column chromatography (SiO2,
hexanes:EtOAc, 85:15 to 75:25) to furnish alcohol 2 (862 mg,
6.9 mmol) as an orange-brown oil in 53% yield. Rf 0.42 (SiO2,
hexanes/EtOAc 60:40). 1H NMR (250 MHz, CDCl3): δ 1.67-1.85
(m, 5H), 1.96 (s, 1H), 2.38 (td, J = 6.9, 1.7 Hz, 2H), 3.72 (t, J =
Phosphonate 3 (278 mg, 1.1 mmol, 1.3 eq.) was placed in a
round-bottom flask with a stirrer bar, the atmosphere was
exchanged for dry N2 and anhydrous THF (10 mL) was added.
Next, the solution was cooled to 0 °C, and NaH in mineral oil

7
(60% w/w, 170 mg, 4.2 mmol, 5 eq.) was added by removing the
HRMS (ESI+): m/z calculated for C14H22NO+ [M+H+]
220.1696, found 220.1710.
ACCEPTED MANUSCRIPT
septum and adding the solid quickly. Then, crude aldehyde (from
Swern step) was added to the reaction as a solution in THF (2 mL
+ 2 mL of washing), the cooling bath was removed, and the
reaction was stirred for 30 min. The reaction was quenched by
addition of aqueous HCl solution (1 M, 10 mL), the mixture was
extracted with EtOAc (15 mL), and the organic phase was
washed with brine (15 mL), dried (MgSO4) and concentrated
under reduced pressure. The product was purified by flash
chromatography (SiO2, hexanes:EtOAc, 75:25) to furnish
spilanthol (1, 118 mg, 0.53 mmol) as a colorless oil in 63% yield.
Rf 0.20 (SiO2, hexanes/EtOAc 75:25). FTIR (ATR, cm-1): 3280,
2957, 2927, 2870, 1668, 1627, 1545, 1235, 1159, 978, 944, 818,
4.2. Biological assays
All reagents and solvents were analytical grade. A eutectic
mixture of 2.5% lidocaine and 2.5% prilocaine (5% EMLA®
cream) was obtained from AstraZeneca (São Paulo, Brazil).
4.2.1. Bioadhesive film production
Hydroxyethyl cellulose (2.5 g) was added to distilled water
(100 mL) under heating (55 ± 2 °C) and stirring, until a
translucent gel was obtained. Agitation was stopped until the
mixture reached room temperature. Next, Tween® 80 (0.7 g),
glycerin (0.7 g) and Transcutol® (2.5 g) were added and
homogenized. Spilanthol (1, 4.14 mg, 18.7 ꢀmol) or analogue 8
(4.12 mg, 18.8 ꢀmol) were diluted with methanol (maximum of
0.5 mL) and the solutions were added to a portion of 30 g of gel.
The resulting mixtures were put on Nylon molds, and these films
were dried for 24 h at 40 °C. Then, the films were cut with a
circular punch (17 mm of diameter).
1
618. H NMR (600 MHz, CDCl3): δ 0.89 (d, J = 6.8 Hz, 6H),
1.70 (d, J = 6.4 Hz, 3H), 1.70-1.81 (m, 1H), 2.23 (q, J = 7.2 Hz,
2H), 2.29 (q, J = 7.2 Hz, 2H), 3.12 (t, J = 6.3 Hz, 2H), 5.23 (dt, J
= 10.3, 7.5 Hz, 1H), 5.67 (dq, J = 14.7, 7.0 Hz, 1H), 5.81 (d, J =
15.2, 1H), 5.78-5.88 (m, 1H), 5.94 (t, J = 10.9, 6.8 Hz, 1H), 6.26
(dd, J = 13.4, 12.6 Hz, 1H), 6.80 (dt, J = 15.3, 7.0 Hz, 1H). 13C
NMR (151 MHz, CDCl3): δ 18.3 (CH3), 20.1 (2CH3), 26.4 (CH2),
28.6 (CH), 32.1 (CH2), 46.9 (CH2), 124.2 (CH), 126.7 (CH),
127.6 (CH), 129.4 (CH), 129.9 (CH), 143.5 (CH), 166.1 (C).
HRMS (ESI+): m/z calculated for C14H24NO+ [M+H+] 222.1852,
found 222.1846.
4.2.2. In vitro permeation studies
In vitro permeation studies were carried out using jacketed
Franz-type vertical diffusion cells (Manual Diffusion Test
System l, Hanson Research Corporation, Chatsworth, CA, USA)
with a permeation area of 1.77 cm2 and a receptor compartment
volume of 7 mL. The jacket was coupled to a water bath (Fisher
Scientific®) at 32 °C. Dermatomed pig ear skin (Nouvag AG),
obtained from a local slaughterhouse (Frigorífico Angelelli,
located in Piracicaba, SP, Brazil, 22°40'09.2"S 47°40'26.6"W)
was used as the barrier in all experiments. The skin was excised
from the outer part of the ear, separated from the underlying
cartilage using a scalpel, dermatomed up to 500 µm thick, and
frozen at −20ꢁ°C until used.55,60 Bioadhesive and skin were
clamped between the donor and receptor compartments. The
bioadhesives were applied in finite dose conditions,, saline-
methanol (70:30, v/v) solution was used in the receptor
compartment to ensure Sink conditions and maintained under
constant magnetic stirring (350 rpm). Samples (400 µL) were
periodically withdrawn from the receptor compartment and
immediately replaced by the same volume of solution, taking
account of dilution effects.
4.1.5. (2E,8E)-N-isobutyldeca-2,8-dien-6-ynamide
(8)
A round-bottomed flask under a N2 atmosphere was charged
with dry CH2Cl2 (10 mL) and oxalyl chloride (169 ꢀL, 2.0 mmol,
2 eq.). The solution was cooled to −78 °C, and anhydrous DMSO
(213 ꢀL, 3.0 mmol, 3 eq.) was added (evolution of gas was
observed). The reaction was stirred for 15 min, then alcohol 2
(124 mg, 1.0 mmol, 1 eq.) was added to the reaction as a solution
in CH2Cl2 (2 mL + 2 mL of washing). The resulting solution was
stirred for 1 h at −78 °C. Next, dry Et3N (836 ꢀL, 6.0 mmol, 6
eq.) was added, the cooling bath was removed, and the reaction
was further stirred for 30 min. The reaction was diluted with
Et2O (20 mL), and the organic phase was extracted with H2O (2 x
10 mL), brine (10 mL), dried (MgSO4), and concentrated in
vacuo (120 mbar, 25 °C, due the volatility of aldehyde). The
product was used in the HWE step without further purification.
Phosphonate 3 (327 mg, 1.3 mmol, 1.3 eq.) was placed in a
round-bottomed flask with a stirrer bar, the atmosphere was
exchanged for dry N2 and anhydrous THF (10 mL) was added.
Next, the solution was cooled to 0 °C, and NaH in mineral oil
(60% w/w, 200 mg, 5 mmol, 5 eq.) was added by removing the
septum and adding the solid quickly. Then crude aldehyde
obtained was added to the reaction as a solution in THF (2 mL +
2 mL of washing), the cooling bath was removed, and the
reaction was stirred for 30 min. The reaction was quenched by
addition of NH4Cl saturated solution (15 mL), the mixture was
extracted with EtOAc (15 mL), and the organic phase was
washed with brine (15 mL), dried (MgSO4) and concentrated
under reduced pressure. The crude product was purified by flash
column chromatography (SiO2, hexanes:EtOAc, 75:25 to 60:40)
to furnish the amide 8 (112 mg, 0.51 mmol) as a white solid in
51% yield. Rf 0.15 (SiO2, hexanes/EtOAc 75:25). FTIR (ATR,
cm-1): 3291, 2959, 2914, 2870, 1666, 1625, 1545, 1334, 1236,
The samples were transferred to chromatography vials for
quantification of spilanthol and analogue by HPLC analysis using
a Waters Alliance HPLC-DAD system (Waters Corp., Milford,
MA, USA) operating with an XTerra MS C18 column (150 mm
x 2.1 mm i.d., 5 µm; Waters) at 40 °C. The mobile phase
consisted of acetonitrile/water 40:60 v/v, with a flow rate of 0.3
mL/min. Absorbance was monitored at 232 nm. The
quantification of spilanthol and analogue 8 was conducted using
external calibration curve with 6 points at a concentration range
of 2 to 50 µg.mL-1, using methanol as diluent. The volume of
injection was 20 µL. All solutions were filtered through a 0.45
µm PVDF membrane before analysis.
The cumulative amount of spilanthol or analogue
8
transported across the skin was plotted as a function of time.
Their flux across the barrier was calculated from the slope of the
linear portion of the curve and the lag time was obtained from the
intercept with the time axis.
1
1220, 950, 650. H NMR (250 MHz, CDCl3): δ 0.87 (d, J = 6.6
Hz, 6H), 1.70 (dd, J = 6.3, 1.6 Hz, 3H), 1.70-1.84 (m, 1H), 2.30-
2.45 (m, 4H), 3.09 (t, J = 6.3 Hz, 2H), 5.43 (dq, J = 15.8, 1.7 Hz,
1H), 5.85 (d, J = 15.3, 1.7 Hz, 1H), 6.00 (dq, J = 15.6, 6.8 Hz,
1H), 5.96-6.12 (m, 1H), 6.69-6.84 (dt, J = 15.3, 6.4 Hz, 1H). 13C
NMR (62.9 MHz, CDCl3): δ 18.3 (CH3), 18.4 (CH2), 20.0
(2CH3), 28.4 (CH), 31.2 (CH2), 46.8 (CH2), 79.9 (C), 86.6 (C),
110.7 (CH), 124.7 (CH), 138.4 (CH), 141.8 (CH), 165.8 (C).
4.2.3. Anesthetic effect evaluation in vivo
Swiss male mice (25-40 g) from Multidisciplinary Center of
Biological Investigation of Laboratory Animals (CEMIB-
University of Campinas) were maintained at 22 ± 2 °C under
light/dark cycles of 12 h. Approval for these assays was provided
by the Animal Ethics Committee of University of Campinas

8
Tetrahedron
ACCEPTED MANUSCRIPT
10. Sharma, V.; Boonen, J.; Chauhan, N. S.; Thakur, M.; De
(protocol #4137-1). The mice were divided into groups of 6
Spiegeleer, B.; Dixit, V. K. Phytomedicine. 2011, 18, 1161-1169.
animals.
11. da Rocha, C. F.; Lima, Y. M. S.; Carvalho, H. O.; Pinto, R. C.;
Ferreira, I. M.; Castro, A. N.; Lima, C. S. Carvalho, J. C. T. J.
Ethnopharmacol. 2018, 214, 301-308.
Topical anesthetic efficacy of bioadhesive films containing
spilanthol (1) or analogue 8 was evaluated with the tail flick
assay. The mouse was placed in an acrylic restraint while the
distal portion of its tail (10 cm) was maintained free (see
Supporting Information). The tail was submitted to irradiation
from an incandescent lamp (55 °C), and the time necessary for
tail removal (latency) was considered as an aversive response to
heat. The baseline was recorded for each animal before the
beginning of the experiment, and only those with baselines below
6 s were considered. In order to avoid tissue damage, the
maximal time for irradiation of the tail was established as 10 s
(cut-off value). Bioadhesive films containing spilanthol (1) or
analogue 8 were compared to EMLA® (150 mg/animal, 7.5 mg of
anesthetic), as a positive control, and the bioadhesive film
without anesthetic, as a negative control. The films or EMLA®
were applied at 2 cm from the tail base for 5 min. The tested
substances were removed, and the nociceptive stimulus was
applied to the same region immediately, and then every 15 min
until the animal returned to its baseline pain response. The
duration of analgesia was defined as the increase in required time
for withdrawal of the tail, which was at least 50% higher than the
baseline value. The data were reported as the percentage of the
maximum possible effect (MPE, in minutes), which was
calculated from the following relation.
12. Wu, L.C.; Fan, N.C.; Lin, M.H.; Chu, I. R.; Huang, S.J.; Hu, C.Y.;
Han, S. Y. J. Agric. Food Chem. 2008, 56, 2341-2349.
13. Escobedo-Martínez, C.; Guzmán-Gutiérrez, S. L.; Hernández-
Méndez, M. M.; Cassani, J.; Trujillo-Valdivia, A.; Orozco-
Castellanos, L. M. Enríquez, R. G. Rev. Bras. Farmacogn. 2017,
27, 214-219.
14. Nomura, E. C.; Rodrigues, M. R.; da Silva, C. F.; Hamm, L. A.;
Nascimento, A. M.; de Souza, L. M.; Cipriani, T. R.; Baggio, C.
H.; Werner, M. F. J. Ethnopharmacol. 2013, 150, 583-589.
15. Cilia-López, V. G.; Juárez-Flores, B. I.; Aguirre-Rivera, J. R.;
Reyes-Agüero, J. A. Pharm. Biol. 2010, 48, 195-200.
16. Cantisani, C.; Macaluso, L.; Frascani, F.; Paolino, G.; D'Andrea
V.; Richetta, A. G.; Calvieri, S. Recent Pat. Inflamm. Allergy
Drug Discov. 2014, 8, 125-131.
17. Kumar, M.; Chawla, R.; Goyal, M. J. Anaesthesiol Clin.
Pharmacol. 2015, 31, 450-456.
18. Daneshkazemi, A.; Abrisham, S. M.; Daneshkazemi, P.; Davoudi,
A. Anesth. Essays Res. 2016, 10, 383-387.
19. Alster, T. Pain Ther. 2013, 2, 11-19.
20. Malamed, S. F. Handbook of Local Anesthesia. 5th ed. Elsevier,
2004.
21. Marra, D. E.; Darwin, Y.; Fincher, E. F.; Moy, R. L. Arch.
Dermatol. 2006, 142, 1024-1026.
22. Hasan, B.; Asif, T.; Hasan, M. Cureus 2017, 9, e1275.
23. FEMA. Safety Assessment of Jambu Oleoresin #3783.
Washington, D.C.: FEMA; 2000.
24. Materials EPoFC. Scientific Opinion on Flavouring Group
Evaluation 303, Revision 1 (FGE.303Rev1): Spilanthol from
chemical group 30. EFSA Journal. 2015, 13.
25. de Freitas-Blanco, V. S.; Franz-Montan, M.; Groppo, F. C.; de
Carvalho, J. E.; Figueira, G. M.; Serpe, L.; Sousa, I. M. O.;
Damasio, V. A. G.; Yamane, L. T.; de Paula, E.; Rodrigues, A. F.
PLoS One 2016, 11, e0162850.
26. Yamane, L. T.; de Paula, E.; Jorge, M. P.; de Freitas-Blanco, V.
S.; Junior, Í. M.; Figueira, G. M.; Anholeto, L. A.; de Oliveira, P.
R.; Rodrigues, R. A. Evid. Based Complement. Alternat. Med.
2016, 2016, 3606820.
27. Gerber, E. Arch. Pharm. 1903, 241, 270-289.
28. Crombie, L.; Krasinski, A. H. A.; Manzoor-i-Khuda, M. J. Chem.
Soc. 1963, 4970-4976.
%MPE = [(test latency − baseline latency)/(cut-off time −
baseline latency) x 100], area under the curve was recorded for
each experimental group.
Competing interests
The authors declare the following competing financial
interests: two patents have been filled on the synthetic route
of spilanthol and analogues (BR1020160178711) and on an
anesthetic formulation and its use (BR10201701137).
29. Ikeda, Y.; Ukai, J.; Ikeda, N.; Yamamoto, H. Tetrahedron Lett.
1984, 25, 5177-5180.
Acknowledgments
30. Ikeda, Y.; Ukai, J.; Ikeda, N.; Yamamoto, H. Tetrahedron 1987,
43, 731-741.
31. Tanaka, S.; Yagi, K.; Ujihara, H.; Ishida, K. WO2009/091040A1.
32. Lombardo, L.; Lankin, M. E.; Ishida, K.; Tanaka, S.; Ujihara, H.;
Yagi, K.; Mei, J. B.; Green, C. B.; Mankoo, A. S.
US2010/0184863A1.
33. Tanaka, S.; Yagi, K.; Ujihara, H.; Ishida, K. US2011/0105773A1.
34. Tanaka, S.; Yagi, K.; Ujihara, H.; Ishida, K. WO2011/007807A1.
35. Tanaka, S.; Ishida, K.; Yagi, K.; Ujihara, H. US2012/0116116A1.
36. Batesky, D. C.; Goldfogel, M. J.; Weix, D. J. J. Org. Chem. 2017,
82, 9931-9936.
The authors acknowledge financial support from the São
Paulo Research Foundation - FAPESP (Awards No. 2014/26378-
2, 2014/25770-6, 2014/16186-9, 2014/06461-2 and 2015/08199-
6), CNPq (award No. 453862/2014-4), and CAPES. We are also
grateful to Renan Galaverna and Prof. Marcos Eberlin
(University of Campinas, Brazil) for HRMS analysis. This work
is dedicated in memory of Sir Derek Barton on occasion of his
centennial birthday.
37. Okuro, K.; Furuune, M.; Enna, M.; Miura, M.; Nomura, M. J.
Org. Chem. 1993, 58, 4716-4721.
38. Feuerstein, M.; Chahen, L.; Doucet, H.; Santelli, M. Tetrahedron
2006, 62, 112-120.
39. Hayashi, T.; Kabeta, K.; Hamachi, I.; Kumada, M. Tetrahedron
Lett. 1983, 24, 2865-2868.
40. Aoki, K. WO2012073910A1.
41. Landor, P. D.; Landor, S. R.; Odyek, O. J. Chem. Soc., Perkin
Trans. 1, 1977, 93-95.
42. Matovic, N. J.; Hayest, P. Y.; Penman, K.; Lehmann, R. P.; De
Voss, J. J. J. Org. Chem. 2011, 76, 4467-4481.
43. Gupta, N.; Kaur, M.; Shallu; Gupta, N.; Kad, G. L.; Singh, J. Nat.
Prod. Res. 2013, 27, 548-553.
44. Kim, S.; Lim, C.; Lee, S.; Lee, S.; Cho, H.; Lee, J.-Y.; Shim, D.
S.; Park, H. D.; Kim, S. ACS Comb. Sci. 2013¸ 15, 208-215.
45. Morimoto, H.; Fujiwara, R.; Shimizu, Y.; Morisaki, K.; Ohshima,
T. Org. Lett. 2014, 16, 2018-2021.
References and notes
1. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629-661.
2. Chung, K.-F.; Kono, Y.; Wang, C.-M.; Peng, C.-I. Bot. Stud.
2008, 49, 73-82.
3. Prachayasittukul, V.; Prachayasittukul, S.; Ruchirawat, S.
Prachayasittukul, V. EXCLI J. 2013, 12, 291-312.
4. Prices from Toronto Research Chemicals and Santa Cruz
Biotechnology in February of 2018.
5. Barbosa, A. F.; de Carvalho, M. G.; Smith, R. E.; Sabaa-Srur, A.
U. O. Rev. Bras. Farmacogn. 2016, 26, 128-133.
6. Chakraborty, A.; Devi, B.R.; Sanjebam, R.; Khumbong, S.;
Thokchom, I.S. Indian J. Pharmacol. 2010, 42, 277-279.
7. Yadav, R.; Yadav, N.; Kharya, M. D.; Savadi, R. Int. J. Pharm.
Pharm. Sci. 2011, 3, 245-247.
8. Gerbino, A.; Schena, G.; Milano, S.; Milella, L.; Barbosa, A. F.;
Armentano, F.; Procino, G.; Svelto, M.; Carmosino, M. PLoS One.
2016, 11, e0156021.
46. For a good review about Sonogashira reaction, see: Chinchilla, R.;
Nájera, C. Chem. Soc. Rev. 2011, 40, 5084-5121.
47. Mohamed, Y. M. A.; Hansen, T. V. Tetrahedron, 2013, 69, 3872-
3877.
9. Spelman, K.; Depoix, D.; McCray, M.; Mouray, E.; Grellier, P.
Phytother. Res. 2011, 25, 1098-1101.

9
48. OECD. Test guideline 428: Skin absorption: in vitro method.
as a plasticizer, Tween® 80 was used as an emulsifier and
ACCEPTED MANUSCRIPT
2004.
Transcutol® was added to facilitate the penetration of active skin.
58. Franz-Montan, M.; Ribeiro, L. N. M.; Volpato, M. C.; Cereda, C.
M. S.; Groppo, F. C.; Tofoli, G. R.; de Araújo, D. R.; Santi, P.;
Padula, C.; de Paula, E. Expert Opin. Drug Deliv. 2017, 14, 673-
684.
59. Jorge, L. L.; Feres, C. C.; Teles, V. E. J. Pain Res. 2010, 4, 11-24.
60. Cereda, C. M. S.; Franz-Montan, M.; da Silva, C. M. G.; Casadei,
B. R.; Domingues, C. C.; Tofoli, G. R.; de Araujo, D. R.; de
Paula, E. J. Liposome Res. 2013, 23, 228-234.
49. Summerfield, A.; Meurens, F.; Ricklin, M. E. Mol. Immunol.
2015, 66, 14-21.
50. Boonem, J.; Baert, B.; Burvenich, C.; Blondeel, P.; Saeger, S. D.;
Spiegeleer, B. D. J. Pharm. Biomed. Anal. 2010, 53, 243-249.
51. Boonen, J.; Baert, B.; Roche, N.; Burvenich, C.; De Spiegeleer, B.
J. Ethnopharmacol. 2010, 127, 77-84.
52. De Spiegeleer, B.; Boonen, J.; Malysheva, S. V.; Mavungu, J. D.;
De Saeger, S.; Roche, N.; Blondeel, P.; Taevernier, L.; Veryser, L.
J. Ethnopharmacol. 2013, 148, 117-125.
53. D’Amour, F. E.; Smith, D. L. J. Pharmacol. Exp. Ther. 1941, 72,
74-79.
Supplementary Material
54. Basha, M.; Abd El-Alim, S. H.; Kassem, A. A.; El Awdan, S.;
Awad, G. Curr. Drug Deliv. 2015, 12, 680-692.
55. de Araujo, D. R.; Padula, C.; Cereda, C. M. S.; Tófoli, G. R.; Brito
Jr., R. B.; de Paula, E.; Nicoli, S.; Santi, P. Pharm. Res. 2010, 27,
1677-1686.
1H, DEPT135 and 13C NMR spectra for all synthesized
compounds. Details for bioadhesive films preparation, in vitro
permeation studies and tai flick assay. Supplementary data
associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tetxxxxxxxxx.
56. Ranade, S.; Bajaj, A.; Londhe, V.; Babul, N.; Kao, D. Eur. J.
Pharm. Sci. 2017, 100, 132-141.
57. Adjuvants were used to improve the properties of the films and to
facilitate the solubilization of the compounds. Glycerin was used