Design and synthesis of conformationally restricted capsaicin analogues based in the 1, 3, 4-thiadiazoleheterocycle reveal a novel family of transient receptor potential vanilloid 1 (TRPV1) antagonists

Article abstract of DOI:10.1016/j.ejmech.2013.05.001

4-hydroxy-3-methoxybenzaldehyde was used as starting material to obtain a number of 1, 3, 4-thiadiazole alkylamide derivatives. The pharmacological properties of these conformationally restricted capsaicin analogues were evaluated on HEK-293T cells transiently expressing TRPV1 receptor. By means of a highthroughput calcium imaging assay we find that 1, 3, 4-thiadiazoles (compounds 8-15) act as potent antagonists of the capsaicin receptor, inhibiting both, the capsaicin- and temperature-dependent activation. Docking studies suggested a different binding orientation on the vanilloid binding site when compared with capsaicin analogues, such as 5-iodononivamide. Overall, our studies suggest that 1, 3, 4-thiadiazoles interact with capsaicin 's binding region of the receptor, although using a different set of interactions within the vanilloid binding pocket.

Full text of DOI:10.1016/j.ejmech.2013.05.001

Accepted Manuscript
Design and synthesis of conformationally restricted capsaicin analogues based in
the 1, 3, 4-thiadiazole heterocycle reveal a novel family of transient receptor potential
vanilloid 1 (TRPV1) antagonists
Carolyne Lespay Rebolledo, Pamela Sotelo Hitshfeld, Sebastiá n Brauchi, Miguel
Zá rraga O.
PII:
S0223-5234(13)00299-7
10.1016/j.ejmech.2013.05.001
EJMECH 6177
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2012
Revised Date: 12 April 2013
Accepted Date: 1 May 2013
Please cite this article as: C.L. Rebolledo, P.S. Hitshfeld, S. Brauchi, M. Zá rraga O., Design and
synthesis of conformationally restricted capsaicin analogues based in the 1, 3, 4-thiadiazole heterocycle
reveal a novel family of transient receptor potential vanilloid 1 (TRPV1) antagonists, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.001.
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Design and synthesis of conformationally
restricted capsaicin analogues based in the
1, 3, 4-thiadiazole heterocycle reveal a novel
family of transient receptor potential
vanilloid 1 (TRPV1) antagonists.
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Carolyne Lespay Rebolledo, Pamela Sotelo Hitshfeld,
Sebastián Brauchi and Miguel Zárraga O.

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European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Design and synthesis of conformationally restricted capsaicin analogues based in
the 1, 3, 4-thiadiazole heterocycle reveal a novel family of transient receptor
potential vanilloid 1 (TRPV1) antagonists
Carolyne Lespay Rebolledoa, Pamela Sotelo Hitshfeldb, Sebastián Brauchib and Miguel Zárraga O.a,
aDepartment of Organic Chemistry, Faculty of Chemical Sciences, University of Concepcion, Edmundo Larenas 160C, Concepción, 1430000, Chile.
bInstitute of Physiology, Faculty of Medicine, University Austral Of Chile, Campus Isla Teja 511-0566,Valdivia, 5090000, Chile.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
4-hydroxy-3-methoxybenzaldehyde was used as starting material to obtain a number of 1, 3, 4-
thiadiazole alkylamide derivatives. The pharmacological properties of these conformationally
restricted capsaicin analogues were evaluated on HEK-293T cells transiently expressing TRPV1
receptor. By means of a high-throughput calcium imaging assay we find that 1, 3, 4-thiadiazoles
(compounds 8-15) act as potent antagonists of the capsaicin receptor, inhibiting both, the
capsaicin- and temperature- dependent activation. Docking studies suggested a different binding
orientation on the vanilloid binding site when compared with capsaicin analogues, such as 5-
iodononivamide. Overall, our studies suggest that 1, 3, 4 thiadiazoles interact with capsaicin’s
binding region of the receptor, although using a different set of interactions within the vanilloid
binding pocket.
Keywords:
TRPV1 receptor
Restriction conformational
Capsaicin analogues
1, 3, 4-thiadiazole
Antagonist activity
.
2012 Elsevier Ltd. All rights reserved.
———
Corresponding author. Tel.: +56-41-2203921; fax: +56-41-2245974; e-mail: mzarraga@udec.cl

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1. Introduction
heterocycle and the effect of conformational restriction between
regions A and B for these capsaicin analogues, which have not
been evaluated before through of the substitution by
heterocyclic group. Thus, from 4-hydroxy-3-
Transient receptor potential vanilloid 1 (TRPV1) proteins are
non-selective, ligand-gated cation channels with permeability to
calcium ions. They are well described as polymodal nociceptors
allowing for the integration of multiple stimulus during the
activation process. These include capsaicin, pH, PIP2, Tº> 43,
and voltage (1, 2, 3). TRPV1 receptors have a direct role in the
physiology and physiopathology of the pain responses showing a
strong correlation with their tissue expression, which includes
peripheral terminals as well as specific brain areas related with
the nociceptive process (4). Thereby, TRPV1 receptors represent
an important pharmacological target, which already have
provided a broad diversity of drugs engineered for the treatment
of inflammatory chronic and neuropathic pain (5, 6).
a
methoxybenzaldehyde as starting material, were carried out
several steps of synthesis, which allowed obtaining 1, 3, 4-
thiadiazole alkylamides (8-15) as novel conformationally-
restricted capsaicin analogues. These analogues included both
iodinated derivatives on the fifth position of vanillyl group and
non-iodinated derivatives, in order to evaluate the effect induced
by iodine atom on this kind of derivatives, due to the differences
of agonist effect and antagonist effect, observed between
compounds non-iodinated and iodinated, respectively on TRPV1
receptors (17, 14). So, as a consequence of the high polarity and
the conformational restriction induced by 1, 3, 4-thiadiazole
heterocycle, together with the effect of the amide group as linker
Capsaicin was the first TRPV1 agonist described, even
though its effects, such as burning pain, increment of the
intracellular calcium and depolarization of DRG sensory neurons,
and potential analgesic effects, were known long before to the
identification of the receptor (7, 8). Structurally, capsaicin has
been an important lead for drug development targeting TRPV1
receptors, leading to the synthesis and identification of important
analogues with well described modifications (9-15). This has
allowed the identification of the minimal structural requirements
essential to modulate TRPV1 activity. According to this, both
capsaicin and capsaicin-like compounds can be divided into three
pharmacophoric structural features (Figure 1). These correspond
to a region A with capacity of forming hydrogen bonds (9); a
polar region B (10), and a lipophilic region C (11). Modifications
performed on these regions have allowed the identification of a
great diversity of compounds with antagonist activity (9-22).
Within the modifications described so far, stand out the
antagonist effect induced by iodine atom in capsaicin analogues,
such as 5-iodononivamide or 6-iodononivamide (25). Likewise,
modifications in the region B such as the substitution of the
amide group by the urea or thiourea group notably increased the
agonist or antagonist activity in both capsaicin analogues as in
non-capsaicin like compounds (10, 16).
group were obtained
antagonists.
a novel family of potent TRPV1
Figure 1. Pharmacophoric features for capsaicin and design of
conformationally restricted capsaicin analogues.
2. Results and Discussion
2.1. Chemistry
In order to increase the antagonist potency, several
optimization strategies have been incorporated in TRPV1
compounds, in which the conformational restriction of linker
groups has shown to be a useful way for this purpose. The
conformational restriction has been incorporated mainly in the
linker group located between regions B and C, through of the
substitution by polar heterocyclic groups, such as pyrimidine
(17), oxazol (18), thiazol (19) among other groups, achieving
potent compounds known as conformationally-restricted TRPV1
antagonist. However, the linker group between regions A and B
corresponding to methylene or amino groups, has been conserved
in these kind of antagonists because the absence or the
substitution by non-flexible groups, such as carbonyl group has
shown to cause lost or decreasing of the activity on TRPV1
receptors (17-22). Although it is true, that many compounds with
restriction conformational have been reported as potent
antagonist TRPV1 and several modifications have been studied
for expand the three pharmacophoric areas mentioned before, the
changes on the linker group between regions A and B through
conformational restriction with heterocyclic groups is little
frequent.
The 1, 3, 4-thiadiazole alkylamide derivatives (8-15) were
synthetized from 4-hydroxy-3-methoxybenzaldehyde in three
steps. Previously, 4-hydroxy-3-methoxybenzaldehyde was
iodinated (scheme 1-I) through in situ oxidation of potassium
iodide with commercial hypochlorite (4.7%). This iodination
method allowed obtaining the monoiodinated product (1), with a
yield of 72%.
According to scheme 1-II the first step consisted in the
synthesis of heterocycle 1, 3, 4-thiadiazole through oxidative
cyclization of the thiosemicarbazones 4 and 5 with an aqueous
solution of ferric chloride. Thus, 2-amino-1, 3, 4-thiadiazole 6
and 7 were obtained with good yields that corresponded to 60%
and 45% respectively. The next step, involved the formation of
amides between 2-amino-1, 3, 4-thiadiazole 6 or 7 and acyl
derivatives coming from linear aliphatic carboxylic acids with
chains of different longitude. According to scheme 2, the
carboxylic acids were converted to acyl chlorides with thionyl
chloride. Later, in situ and at room temperature the 2-amino-1, 3,
4-thiadiazole 6 or 7 was added into the reaction medium followed
by the addition of pyridine. The esterified amide derivatives were
obtained by this method, therefore the last step of synthesis
consisted in an alkaline hydrolysis reaction with NaOH 0.1 M
under reflux at 60°C approximately for achieve the formation of
a medium of reaction homogenous. This method led to obtaining
Thereby, in this work have been proposed the design of
conformationally-restricted capsaicin analogues by replacing the
amide group of capsaicin with the 1, 3, 4-thiadiazole heterocycle,
followed by the incorporation of an amide group as linker group
between regions B and C (Figure 1). This isosteric replacement
has allowed evaluating both the isosteric efficiency of this

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Scheme 1. Reaction Conditions: (a) methanol, water-ice bath, agitation for 1 hour (b) room temperature, H2SO4 (c) agitation for 1
hour, methanol, glacial acetic acid, pH 4-5, reflux for 2 hours (d) methanol, reflux for 1-2 hours.
Scheme 2. Reaction Conditions: (a) Reflux at 80º C for 16 hours, (b) Agitation for 3 hours at room temperature and (c) Methanol for
3 hours at 60°C.
the 1, 3, 4-thiadiazole alkylamide derivatives 8-15, which were
purified by chromatography and crystallization, achieving
satisfactory yields of synthesis.
2.2. Biological Evaluation
All the 1, 3, 4-thiadiazole derivatives obtained were tested in
transiently transfected HEK-293T cells expressing rat TRPV1
(rTRPV1). The biological activity of the derivatives was
evaluated on the activation of the rTRPV1 receptor by

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temperature and capsaicin. TRPV1 activation was measured by
increased the total response (Supporting information, Figure
1), as expected according to the allosteric potentiation reported
for TRPV1 channels (Latorre et al. 2009). The effect of 1, 3, 4-
thiadiazole derivatives (8-15) on the activity of rTRPV1 was
therefore evaluated at 50°C. As seen in Figure 2b, the cells
incubated in the presence of thiadiazole derivatives displayed a
severely diminished calcium influx signal when compared with
means of a high-throughput calcium influx assay; following the
fluorescence of Fluo-4 AM loaded cells in a real time PCR
thermocycler (23). This method allowed us to follow multiple
conditions simultaneously during a temperature ramp as well as
to maintain controlled temperatures.
First, the response to temperature changes for control
conditions was evaluated from 22°C to 56 °C. These results are
shown in Figure 2a in which the temperature-induced calcium
influx was normalized with respect to the fluorescence obtained
at 50°C for rTRPV1. The temperature activation curve for
rTRPV1 receptors rose around 40ºC and it saturated at 54º C. The
calcium influx induced by increasing temperature was modest or
not observed in the cultures previously incubated with reported
TRPV1 blockers, such as BCTC, lanthanum, or ruthenium red
(6). In contrast, the incubation in presence of 30 nM capsaicin
control conditions.
Furthermore, was observed that cells
incubated in the presence of thiadiazole derivatives inhibited the
calcium influx evoked by 30 nM of capsaicin (Figure 2c).
According to these results, a low micromolar range (1µM) of the
thiadiazole derivatives was enough to block both capsaicin- and
temperature- dependent activation of rTRPV1 receptors (Table
1).
Using the same approach, dose-response curves were built
and the half maximal inhibitory concentration (IC50) for previous-
Figure 2. Activity of 1, 3, 4-thiadiazole derivatives on rat TRPV1 receptors. (A) Calcium imaging recordings performed on HEK-
293T cell overexpressing rTRPV1. The temperature ramp induced a significant raise in the [Ca2+]i curve over 40°C. The response
after incubations with the TRPV1 blockers Lanthanum (100 ꢀM), Ruthenium Red (10 ꢀM) and BCTC (1.0 ꢀM) and in absence of
them (rTRPV-1) is shown. The changes in fluorescence were estimated by the following correction (F-F0)/F0, and further normalized
with respect to the normal (i.e. untreated transfected cells) response at 50°C (black circles). Data represent mean ± s. d. (n=8). (B)
Bar plot for the response elicited by incubations with 1.0 ꢀM of thiadiazoles and compared with the antagonistic activity of BCTC
(1.0 ꢀM), Lanthanum (100 mM) and Red Ruthenium (10 ꢀM). The relative fluorescence was normalized with respect to the normal
TRPV1 response at 50°C and was represented as mean ± s. d. for two assays performed independently (n=3). (C) Bar plot for the
inhibitory effect of the thiadiazole derivatives (1.0 µM) with respect to the response observed after capsaicin stimulation. The
response was normalized to the [Ca2+]i signal induced by 30 nM capsaicin at 25 °C. The responses correspond to the mean ± s. d.
(n=10). All data analyzed were statistically different ***P < 0.05 (one-way ANOVA).
Table 1. Percentage of inhibition for 1, 3, 4-thiadiazole derivatives evaluated at 1.0 µM on rat TRPV1 receptors
Compound
% Inhibitiona
(Temperature)
92.92 ± 0.88
89.78 ± 0.83
93.95 ±1.31
88.85 ± 0.35
91.86 ± 0.93
93.68 ± 1.42
92.36 ± 0.83
94.29 ± 1.64
66.38 ± 0.32
% Inhibitiona
(Capsaicin)
98.75 ± 0.65
97.72 ± 0.14
95.33 ± 0.25
95.47 ± 0.07
96.92 ± 0.13
94.52 ± 0.18
90.78 ± 0.72
96. 60 ± 0.16
92.66 ± 0.25
IC50 (nM)a
(Capsaicin)
0.18 ± 0.04
0.15 ± 0.04
0.77 ± 0.02
2.61 ± 0.05
0.34 ± 0.04
0.33 ± 0.03
6.07 ± 0.01
1.47 ± 0.03
4.86 ± 0.12
IC50 (nM)a
(Temperature)
0.05 ± 0.01
-
8
9
10
1.26 ± 0.01
-
11
12
0.15 ± 0.05
1.09 ± 0.49
-
13
14
15
0.29 ± 0.03
2.12 ± 0.35
BCTC
a
Values were obtained in two independent experiments and expressed as mean ± s. d.

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Figure 3. Dose-Response Curves obtained for 1, 3, 4-thiadiazole derivatives through capsaicin and temperature-induced activation
on rat TRPV1. The fluorescence values of temperature activation mode were obtained at 50°C to build the curves. The relative
fluorescence was calculated for each concentration and normalized with respect to the maxima fluorescence. Values are expressed as
mean ± s. d. (n=3). Solid traces represent a fit of the data to the Hill equation.
ly tested derivatives was calculated (Figure 3 and Table 1). The
potency determined by dose-response curves confirmed the low
nanomolar order of magnitude necessary for antagonist activity
of 1, 3, 4-thiadiazole derivatives. BCTC was used as control
because its general use as a more specific TRP channels inhibitor
(24). In the performed experiments, the IC50 for BCTC was
determined to be 4.8 nM and 2.1 nM on capsaicin, and
temperature activation respectively. This is in good agreement
with previously reported active concentrations (~5nM; 23).
Compounds 8 and 9 were the most potent among all the
evaluated compounds (IC50= 40pM; Table 1), showing higher
affinity when compared with other antagonists such as iodo-
nonivamide and iodo-resiniferatoxin (25-27).
rocycle is responsible for the differences observed in the potency
of the compounds when compared themselves or to other
capsaicin analogues reported before (15, 25). Furthermore, the
antagonistic effect shown by the derivatives with the non-
iodinated vanillyl group was in opposition to the effect reported
for other capsaicin analogues containing amide, thiourea, or the
1, 2, 3-triazole group as “B region”, all of them elicited a strong
agonistic effect (10, 15).
In general terms, the level of inhibition of capsaicin-induced
activation of rTRPV1 indicate that the non-iodinated derivatives
are better antagonists in comparison with the iodinated
derivatives. Our analysis also underscores the compounds with
longest aliphatic chains (8, 9, 12 and 13) as the most potent. On
the other hand, the inhibitory responses observed for the case of
temperature-induced activation of rTRPV1 showed that
derivatives 8, 12 and 15, having the longest and the shortest
linear aliphatic chains were the most potent. Interestingly, when
the same temperature activation assay was performed on the
capsaicin-insensitive chicken TRPV1 receptor, the derivatives 11
and 15 with the shortest aliphatic chains showed greater potency
and in general the iodinated derivatives showed slightly a better
potency than non-iodinated derivatives (Table 2).
In order to evaluate whether 1, 3, 4-thiadiazole derivatives
share capsaicin's mechanism of action, we further evaluated their
activity in the avian TRPV1 receptors (chicken clone; cTRPV1)
which are insensitive to capsaicin. Although capsaicin
unresponsive,
cTRPV1 receptors are activated by heat or
protons, consistent with their physiological role as nociceptors
(28).
According to our calcium imaging approach, the activation of
the cTRPV1 receptor induced by temperature was absent in
presence of lanthanum, ruthenium red, and all the thiadiazole
derivatives tested (Figure 4b). This time, the effect of BCTC was
modest in comparison with the thiadiazole derivatives (Table 2).
This may imply that the natural substitutions present in cTRPV1
clone are important not only for capsaicin-dependent activation
but also for BCTC-dependent inhibition. Interestingly,
thiadiazole derivatives displayed a lesser but still potent
inhibition of the cTRPV1 temperature-activation when compared
to the response obtained with the rTRPV1 receptor (Table 2).
These results indicated that although the natural structural
variations present in cTRPV1 are not necessary for the molecular
mechanism underlying the effect of thiadiazole derivatives, they
are important in the modulation of the inhibitory effect.
3. SAR analysis, docking and electrostatic potential
Docking studies have shown that vanillyl group of all
vanilloid compounds (e.g. capsaicin) interacts with the same
aminoacids in the vanilloid binding site (29), and mutagenesis
studies have showed that Y511, S512 and T550 residues are
associated with the sensibility to vanilloid compounds (28, 30).
According to the results presented here, the presence of
capsaicin altered the interaction of the thiadiazole derivatives
within the receptor (Figure 2 and Table 1). Additionally,
residues that alter the pathway for capsaicin-dependent activation
also, alter (but not impaired) the effect of the synthesized
derivatives. Therefore, our results suggest to us that to elicit its
antagonistic activity, the thiadiazole derivatives require
pathway of activation that share elements with the one of
capsaicin. These interactions not necessarily have to do with bin-
a
Notably, both families of thiadiazole derivatives tested (i.e.
iodinated and non-iodinated) presented the same trend of
antagonistic activity, suggesting that the 1, 3, 4-thiadiazole hete-

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Figure 4. Temperature-induced activation in chicken TRPV1 for A) conditions control corresponding to cells incubated in presence
of lanthanum (100 mM), ruthenium red (10 ꢀM), BCTC (1.0 ꢀM), capsaicin (30 nM) and in absence of them (cTRPV-1). B) 1, 3, 4-
thiadiazole derivatives evaluated at 1.0 ꢀM. The relative fluorescence was normalized with respect to the response of untreated
transfected cells at 50°C. The relative fluorescence measurements were expressed as mean ± s. d. for two assays performed
independently (n=3). Data obtained for the antagonist effect are statistically different ***P < 0.05 (one-way ANOVA) with the
exception of capsaicin and BCTC.
alkyl group orients toward a hydrophobic pocket formed by Ile
514, Leu 515, Thr 556, Leu 553, and Trp 549 whereas, the
thiadiazole heterocycle and the amide group are oriented near of
the residues of Phe 517, Leu 521, Val 518 and Tyr 511.
Table 2. Antagonist activity of 1, 3, 4-thiadiazoles evaluated
at 1.0 µM on chicken TRPV1 receptors
Compound
% Inhibitiona
79.38 ± 0.30
81.18 ± 5.62
92.56 ± 1.72
80.07 ± 8.13
85.43 ± 0.15
95.29 ± 0.41
8.97 ± 0.89
When we cross correlate these results obtained by molecular
docking with the inhibitory effect of thiadiazole derivatives on
capsaicin-dependent rTRPV1 activation, we can observe that the
most potent compounds are characterized by an interaction
between Thr 550 and the vanillyl group of the ligand through
hydrogen bond formation. This is accompanied by polar
interactions between the thiadiazole group and polar residues of
the receptor. On the other hand, the less potent compounds
interact by hydrogen bond formation between Tyr 511 and the
amide group of thiadiazole derivatives (Figure 5d). When we
analyze the same correlation for the case of the inhibition of the
temperature-induced activation, the docking results indicated that
the vanillyl group of the most potent derivatives interacts with
Thr 550, together with an apparent important contribution from
the polar interactions established by the thiadiazole and amide
groups (Table 3). Therefore, when we compared compounds 8,
12, and 15 our results suggest that Thr 550 promotes a greater
antagonistic potency when compared with Tyr 511 on capsaicin-
induced responses, while polar interactions between thiadiazole
and amide groups promotes greater antagonistic potency on the
temperature-induced response.
8
9
11
12
13
15
BCTC
a
Values were obtained in two independent experiments and
expressed as mean ± s. d.
ding properties, since they may also include the allosteric
communication between the agonist-sensing domain and the
pore.
In order to evaluate these structural features of thiadiazole
derivatives that are responsible for their higher activity with
respect to their non-restricted analogs, and to explore into the
mechanism of protein-ligand interaction, we performed docking
studies on the vanilloid binding site of a rTRPV1 (Gavva 2004).
The docking results for capsaicin (Figure 5a) indicated that the
vanillyl group interacts with Tyr 511 by means of the formation
of hydrogen bonds, whereas the alkyl group is oriented towards a
hydrophobic pocket formed by Val 518, Met 547, Leu 521, Phe
517, Phe 522 and Thr 550. This docked pose is in agreement with
the mutagenesis studies reporting that Tyr 511 and Thr 550 are
associated with the sensibility of TRPV1 receptors to vanilloids
(28, 30). The binding orientation adopted by 5-iodononivamide
was similar to the one observed for capsaicin on the vanilloid
binding site (Figure 5b), and involved the same set of residues.
In contrast, the docking results for 1, 3, 4-thiadiazole derivatives
(Figure 5 and Table 3) showed not only a different binding
orientation into the vanilloid binding site but also a different set
of interactions. According to this, the vanillyl group interacts
with Thr 550 through the formation of hydrogen bonds and the
The differences between capsaicin and thiadiazole derivatives are
extensive to the active conformation of the compounds once on
the binding pocket. On figure 6 we can observe that agonists and
antagonists adopt different three dimensional configurations. We
analyzed then the electrostatic potential for the bioactive
conformation of thiadiazole derivatives (Figure 6). Our analysis
reveals that thiadiazole derivatives possess an electrostatic
potential pattern resembling that of capsaicin and 5-
iodononivamide. According to the pharmacophoric division
mentioned initially for capsaicin, the regions with the highest
electron density were the oxygen of the carbonyl group and the
nitrogen atoms of the thiadiazole group in the region B, and the
oxygen atoms of the hydroxyl and methoxyl groups located in the
region A. Conversely, the lowest electron density was observed in
the aromatic ring of the vanillyl group (region A). Moreover, an
unequal distribution of electron density was observed on the
region C formed by the alkyl group. The latter might mediate Van
der Waals or hydrophobic interactions, which would explain the

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Figure 5. Docking solutions into the vanilloid binding site of TRPV1 receptor for (A) Capsaicin, (B) 5-iodononivamide, (C)
thiadiazole 8 and (D) thiadiazole 13. The interactions by means of hydrogen bonds are shown in green, whereas the residues
involved the formation of hydrogen bonds are shown in blue, the residues involved in the polar interactions are shown in red and
the residues involved in the hydrophobic interactions are shown in black.
Table 3. Summary of docking results for lowest energy poses of capsaicin, 5-iodononvamide, BCTC and 1, 3, 4-thiadiazole
derivatives. The residues involved in the interaction on vanilloid binding site and the nature of interaction are shown. Blue color
indicates the residues forming hydrogen bond, red color indicates the residues involved in the polar interaction and black color
indicates the hydrophobic or Van der Waals interactions.
Compound
Docking Residues
E pose
(Kcal/mol)
-8.14
Tyr 511, Ile 514, Leu 515, Phe 517, Val 518, Leu 521, Phe 522, Val 525,
Met 547, Thr 550, Leu 553, Thr 556
Capsaicin
Tyr 511, Ile 514, Leu 515, Phe 517, Val 518, Gln 519, Leu 521, Phe 522, Val
524, Met 547, Thr 550, Leu 553, Thr 556
Tyr 511, Leu 515, Val 518, Leu 521, Phe 522, Val 525, Phe 543, Met 547,
Thr 550, Leu 553, Tyr 554, Tyr 555, Phe 559
Tyr 511, Ile 514, Leu 515, Phe 517, Val 518, Leu 521, Phe 522, Met 547,
Ala 546, Trp 549, Thr 550, Leu 553, Thr 556
Tyr 511, Ile 514, Leu 515, Val 518, Ala 546, Trp 549, Thr 550, Asn 551,
Met 552, Leu 553, Tyr 555, Thr 556, Phe 559
Val 518, Leu 521, Phe 522, Val 525, Met 547, Trp 549, Thr 550, Leu 553,
Tyr 555, Thr 556, Phe 559
Tyr 511, Ile 514, Leu 515, Phe 517, Val 518, Leu 521, Phe 522, Thr 550,
Leu 553, Met 552, Tyr 554, Tyr555, Thr 556, Phe 559
Tyr 511, Ile 514, Leu 515, Val 518, Leu 521, Trp 549, Thr 550, Met 552,
Leu 553, Met 552, Tyr 555, Thr 556, Phe 559
Tyr 511, Ile 514, Leu 515, Phe 517, Val 518, Leu 521, Trp 549, Thr 550,
Met 552, Leu 553, Met 552, Tyr555, Thr 556, Phe 559
Tyr 511, Ile 514, Leu 515, Phe 517, Val 518, Leu 521, Thr 550, Met 552,
Leu 553, Tyr555, Thr 556, Phe 559
Tyr 511, Leu 515, Val 518, Phe 522, Trp 549, Thr 550, Asn 551, Leu 553,
Tyr554, Tyr 555, Thr 556, Arg 557, Phe 559
-8.33
-8.52
-6.82
-6.76
-6.57
-7.49
-7.01
-7.42
-5.90
-8.55
5-iodononivamide
BCTC
8
12
9
13
10
14
11
15

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Figure 6. Map of electrostatic potential for docked poses of capsaicin, 5-iodononivamide and 1, 3, 4-thiadiazole derivatives. Red
color represent zones with high electron density, Blue color represent zones with low electron density and White color represent zones
with neutral electron density.
greater antagonistic potency obtained when longer alkyl groups
were introduced.
5. Experimental Section
5.1 Chemistry
The results obtained from our in-silico studies, specially the
similarities observed on the electrostatic potential, suggest a
common binding region for capsaicin and thiadiazole derivatives
into the receptor, however, having both a different set of
interactions with the receptor. These differences may be
determined by the structural restrictions intrinsic to the designed
antagonists together with the orientation of the compounds inside
the binding pocket.
Unless otherwise noted all materials and solvents were
obtained from Merck and were used without previous
purification. The reactions involved moisture sensitive reagents
were carried out under presence of CaCl2 anhydride. The
reactions were followed by means of thin layer chromatography
and were revealed by UV light, sulfuric/vanillin spray and Folin
reactive. Preparative column chromatography was performed
using 60G-F254 silica gel (MERCK). The melting points were
determined on a Bûchi oil-heated melting point apparatus and are
uncorrected; the 1H-NMR and 13C-NMR spectra were recorded on
a Bruker Avance-400 MHz (Avance 400, Bruker, Germany,
2002). The FT-IR spectrums were recorded with a Nicolet
Nexus spectrometer (Nexus, Nicolet Instrument. Inc, Madison
WI 53711, USA) and the elemental analysis was performed on
Elemental Analyzer CE Instrument (EA 1108, Fisons, Italy,
1995).
The fact that BCTC showed a reduced activity on cTRPV1 when
compared with the inhibition caused in rTRPV1 well correlate
with our structural studies. The docked conformation of BCTC
inside the vanilloid binding site share similarities with capsaicin
rather than to thiadiazole derivatives (Table 3), causing then a
modest inhibitory effect on channel activity. We speculate that
capsaicin and BCTC are able to bind to cTRPV1, however, the
chemical energy of binding is not transduced to the pore domain,
probably due to the presence of a different set of allosteric
interactions, allowing for thiadiazole inhibition but impairing
capsaicin-dependent activation.
5.1.1 Synthetic Procedures
5.1.1.1. 4-Hydroxy-3-iodo-5-methoxybenzaldehyde (1). In a
100 ml Erlenmeyer flask was added 4-hydroxy-3-
methoxybenzaldehyde (66 mmol, 10 g) and potassium iodide (66
mmol, 9.73 g) in 20 ml of methanol. The reaction mixture was
cooled under an ice-water bath for 15 minutes and after was
added drop to drop, using an addition funnel and under stirring,
74 ml of sodium hypochlorite commercial (66 mmol, 75 ml) and
then the reaction mixture was stirred for 1 hour, maintaining the
ice-water bath. After was added 20 ml of an aqueous solution of
sodium thiosulphate (10% p/v) and stirred for 15 minutes at room
temperature and then acidified with hydrochloric acid (37%) until
pH 3-4, the precipitate formed was filtered and washed with
cooled water. The crude product was crystallized from isopropyl
alcohol and 1 was obtained as light yellow crystals.
4. Conclusion
The conformationally restricted capsaicin analogues 1, 3, 4-
thiadiazole derivatives showed a potent antagonistic activity (low
nanomolar) on capsaicin- and temperature- activated TRPV1
channels. The modification from agonist to antagonist activity,
seems to be associated with conformational effects and an
increased polarity on the region B of the analogues. Furthermore,
our results indicated that the iodinated thiadiazole derivatives are
approximately 100 times more potent antagonists than other
iodinated capsaicin analogues reported in the past (14, 15).
According to our structural studies this greater antagonist
potency observed for thiadiazole derivatives was possible due to
a different binding orientation into the vanilloid binding site,
different to the one observed for other capsaicin derivatives, such
as 5-iodononivamide.
5.1.1.2. 4-Formyl-2-methoxyphenyl acetate (2). In a 100 ml
Erlenmeyer flask was added 4-hydroxy-3-methoxybenzaldehyde
(66 mmol, 10 g) and an excess of acetic anhydride (27 mmol, 30
ml) and then under stirring at room temperature was added 0.3 ml

ACCEPTED MANUSCRIPT
of sulphuric acid concentrated. The reaction mixture was
similar procedure for synthesis of 8. It was used 1ml of nonanoic
acid (11 mmol) and 0.6 ml of thionyl chloride (8.30 mmol). After
was added 200 mg of 7 (0.89 mmol) and 12 was obtained as a
brown powder.
maintained in stirring continued for 4 hours and then slowly was
added cold water until to form a solid, which was filtered and
washed with abundant water. The crude product was crystallized
from methanol to give 2 as light orange-pink crystals.
5.1.1.10. N-[5-(4-hydroxy-3-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] octanamide (9). The product was obtained by
similar procedure for synthesis of 8. It was used 1ml of octanoic
acid (13 mmol) and 0.8 ml of thionyl chloride (11 mmol). And
then 200 mg (0.89 mmol) of 6 was added, obtaining 9 as white
crystals in a 55.27% of yield.
5.1.1.3. 4-Formyl-2-iodo-6-methoxyphenylacetate (3). Similar
procedure for synthesis of 2 was followed. It was used 4-
hydroxy-3-iodo-5-methoxybenzaldehyde (14.40 mmol, 4 g) and
acetic anhydride (212 mmol, 20 ml). The product 3 was obtained
as light brown crystals.
5.1.1.4. 2-Methoxyphenyl acetate thiosemicarbazone (4). In a
250 ml round bottom flask a mixture of thiosemicarbazide (3.73
g, 41 mmol) and 2 (8 g, 41 mmol) in 50 ml of methanol was
prepared and acidified until pH 4-5 with acetic acid glacial and
refluxed for 6 hours under stirring. The reaction mixture was
cooled in an ice-water bath and the precipitate formed was
filtrated and crystallized from methanol to give 4 as light yellow
crystals.
5.1.1.11. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] octanamide (13). Similar procedure for
synthesis of 8 was followed. It was used 1 ml of octanoic acid
(13 mmol) and 0.8 ml of thionyl chloride (11 mmol). And then
200 mg (0.57 mmol) of 7 was added, obtaining 13 as a brown
powder.
5.1.1.12.
N-[5-(4-hydroxy-3-methoxyphenyl)-1,
3,
4-
thiadiazol-2-yl)] heptanamide (10). The product was obtained by
similar procedure for synthesis of 8. It was used 1 ml of
heptanoic acid (14 mmol) and 0.9 ml of thionyl chloride (12
mmol). And then 200 mg (0.89 mmol) of 6 was added, obtaining
10 as white crystals.
5.1.1.5. 2-Iodo-6-methoxyphenylacetate thiosemicarbazone
(5). Similar procedure for synthesis of 4 was followed, were used
thiosemicarbazide (12 mmol, 1.09 g) and 3 (12 mmol, 4 g). The
product 5 was obtained as light yellow crystals.
5.1.1.6. 2- Amino- 5 - (4-hydroxy-3-methoxyphenyl) - 1, 3, 4-
thiadiazole (6). In a 250 ml round bottom flask was added 4 (19.5
mmol, 5g), 20 ml of ferric chloride aqueous solution (59 mmol,
9.48g) and 30 ml methanol. The reaction mixture was refluxed
under stirring for 1-2 hours and filtered in hot on activated
carbon and then an aqueous solution of citric acid (11.52 g, 60
mmol) and sodium citrate (6.42 g, 30 mmol) was added and
refluxed for 1 hour under stirring. After the solution was
neutralized with 10% aqueous ammonia to pH 4-5 and cooled in
an ice- water bath. The precipitate was washed several times with
cooled water and then was crystallized with a mixture of
ethanol/methanol 1:1 to give 6 as a bright yellow powder.
5.1.1.13. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] heptanamide (14). The synthetic procedure was
similar to described for 8. It was used 1 ml (14 mmol) of
heptanoic acid and 0.9 ml of thionyl chloride (12 mmol). And
then 200 mg (0.57 mmol) of 7 was added, obtaining 14 as a
brown powder.
5.1.1.14.
N-[5-(4-hydroxy-3-methoxyphenyl)-1,
3,
4-
thiadiazol-2-yl)] hexanamide (11). The product was obtained by
similar procedure for synthesis of 8. It was used 1 ml of hexanoic
acid (16 mmol) and 0.9 ml of thionyl chloride (12 mmol). And
then 200 mg (0.89 mmol) of 6 was added, obtaining 11 as white
crystals.
5.1.1.7. 2-Amino-5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3,
4-thiadiazole (7). Similar procedure for synthesis of 6 was
followed. It was mixed 5 (10.60 mmol, 4g) and a ferric chloride
aqueous solution (32 mmol, 10 ml, 5.2g,). Finally, the product
was crystallized with a mixture of water/methanol 1:1 to give 7
as a light brown powder.
5.1.1.15. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] hexanamide (15). The synthetic procedure was
similar to described for 8. It was used 1 ml (16 mmol) of
hexanoic acid and 0.9 ml of thionyl chloride (12 mmol). And
then 200 mg (0.57 mmol) of 7 was added, obtaining 15 as a
brown powder.
5.1.1.8. N-[5-(4-hydroxy-3-methoxyphenyl)-1, 3, 4- thiadiazol-
2-yl)] nonamide (8). In a 250 ml round bottom flask dried
previously, was added 1 ml of nonanoic acid (11 mmol) and 0.6
ml of thionyl chloride (8.30 mmol), the reaction mixture was
stirred and refluxed for 16 hours at 80°C . Later, the reaction
mixture was cooled at room temperature and under vigorous
stirring was rapidly added 6 (0.89 mmol, 200 mg) to reaction
medium, followed of 2 ml of pyridine. The mixture reaction was
stirred at room temperature for 2 hours and then partitioned
between an aqueous acid solution and dichloromethane; the
organic layer was extracted and washed with a saturated aqueous
solution of sodium chloride. After the concentrated organic layer
was hydrolyzed in presence 2 ml of a solution sodium hydroxide
0.1 M in 10 ml of methanol and was refluxed for 3 hours under
stirring. The hydrolysis mixture was acidified until pH 4 with
hydrochloric acid 37%, a white precipitate was formed and then
filtered followed of the purification by column chromatography
using 20% ethyl acetate/hexane, and finally the product obtained
was crystallized in 1:4 water / methanol to give 8 as white
crystals.
5.1.2. Characterization of Compounds
5.1.2.1. 4-Hydroxy-3-iodo-5-methoxybenzaldehyde (1). Yield:
75%; mp: 182°C; TLC: Rf = 0.6 (dichloromethane). IR (KBr) ν
cm-1: 1415.48, 1455.92, 1491.49, 1578.21, 1665.52, 2842.86,
3145.56.
5.1.2.2. 4-Formyl-2-methoxyphenyl acetate (2). Yield: 71%;
mp: 66°C; TLC: Rf =: 0.8 (70% ethyl acetate/ether petroleum).
IR (KBr) ν cm-1: 1029.62, 1210.29, 1279.61, 1428.30, 1468.36,
1509.57, 1596.57, 1684.91, 1756.07, 2968.94, 2846.29, 3015.06.
5.1.2.3. 4-Formyl-2-iodo-6-methoxyphenylacetate (3). Yield:
99%; mp: 62°C; TLC: Rf = 0.7 (60% ethyl acetate/ether
petroleum). IR (KBr) ν cm-1: 1203.78, 1275.01, 1418.37,
1461.59, 1579.20, 1685.51, 1768.15, 2966.65, 3051.19.
5.1.2.4. 2-Methoxyphenyl acetate thiosemicarbazone (4).
Yield: 89%; mp: 119°C; TLC: Rf = 0.6 (70% ethyl acetate/ether
petroleum). IR (KBr) ν cm-1: 1029.58, 1214.36, 1294.31,
1368.13, 1464.26, 1509.53, 1538.31, 1595.55, 1762.03, 3014.01,
3160.41, 3263.58, 3395.89.
5.1.1.9. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] nonamide (12). The product was obtained by

ACCEPTED MANUSCRIPT
5.1.2.5. 2-Iodo-6-methoxyphenylacetate thiosemicarbazone
5.1.2.11. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] octanamide (13). Yield: 33.46%; mp: 158°C;
TLC: Rf = 0.28 (60% ethyl acetate/ether petroleum). IR (KBr) ν
(5). Yield: 85%; mp: 184°C; TLC: Rf = 0.63 (80% ethyl
acetate/ether petroleum). IR (KBr) ν cm-1: 1279.48, 1409.29,
1460.59, 1514.49, 1587.62, 1763.25, 2830.59, 2972.53, 3151.73,
3262.22.
1
cm-1: 3386.16, 3386.16, 3158.92, 2929.56-2864.87, 1695.60. H
NMR (400 MHz, DMSO) δ ppm: 0.87-0.84 ( t, J= 6.7 Hz, 1H),
1.28-1.24 (m, 8H), 1.63-1.59 (m, 2H), 2.20-2.16 (t, J= 7.4Hz,
2H), 3.36 (s, 3H),7.48-7.47 (d, J= 1.9 Hz, 1H), 7.78-7.77 (d, J=
1.9 Hz, 1H), 10.04 (s, 1H), 12.57 (s, 1H). 13C NMR (101 MHz,
DMSO) δ ppm: 172.88, 166.65, 158.75, 147.60, 141.94, 137.50,
132.94, 128.57, 74.39, 56.54, 31.45, 28.90, 28.79, 28.70, 24.96,
22.41, 14.30. Molecular Formula: C17H22IN3O3S. Anal. Calcd: C
(42.95), H (4.66), N (8.84), O (10.10), S (6.75), I (26.70). Found:
C (43.03), H (4.52), N (8.91), O (9.53), S (6.53), I (27.48).
5.1.2.6. 2- Amino- 5 - (4-hydroxy-3-methoxyphenyl) - 1, 3, 4-
thiadiazole (6). Yield: 60%; mp: 263°C; TLC: Rf = 0.03 (70%
ethyl acetate/ether petroleum). IR (KBr) ν cm-1: 1282.25,
1370.74, 1423.82, 1511.31, 1580.33, 1642.43, 2784.57, 3094.62,
1
3214.19, 3287.48. H NMR (400 MHz, DMSO) δ ppm: 3.86 (s,
3H), 4.25 (s, 2H), 6.96-6.94 (d, J=8.2 Hz, 1H), 7.19-7.17 (dd, J=
8.2 Hz, 1.9 Hz, 1H), 7.30-7.29 (d, J= 1.8 Hz, 1H), 9.20 (s, 1H,
OH). 13C NMR (101 MHz, DMSO) δ ppm: 168.24, 156.07,
150.05, 148.17, 120.76, 119.82, 115.95, 109.51, 55.77.
5.1.2.12.
N-[5-(4-hydroxy-3-methoxyphenyl)-1,
3,
4-
thiadiazol-2-yl)] heptanamide (10). Yield: 55.48%; mp: 224°C;
TLC: Rf = 0.26 (60% ethyl acetate/ether petroleum). IR (KBr) ν
5.1.2.7. 2-Amino- 5 - (4-hydroxy-3-iodo-5-methoxyphenyl) - 1,
3, 4-thiadiazole (7). Yield: 45%; mp: 281°C; TLC: Rf = 0.23
(80% ethyl acetate/ether petroleum). IR (KBr) ν cm-1: 1413.95,
1487.43, 1586.70, 1637.74, 3293.55; 1H NMR (400 MHz,
DMSO) δ ppm: 3.81 (s, 3H), 5.69 (s, 1H), 7.273- 7.268 (d, J=
2.17 Hz, 1H), 7.282 (s, 2H), 7.469-7.464 (d, J= 1.9 Hz, 1H). 13C
NMR (101 MHz, DMSO) δ ppm: 56.10, 84.57, 109.10, 124.04,
128.14, 147.17, 147.83, 155.03, 168.01.
1
cm-1: 3536. 01, 3212.54, 3091.83, 2930.79-2775.56, 1676.99. H
NMR (400 MHz, DMSO) δ ppm: 0.83-0.87 ( dd, J= 8.9Hz, 4.8
Hz, 3H), 1.32-1.23 (m, 6H), 1.63-1.56 (m, 1H), 2.49-2.46 (t,
J=7.9 Hz, 2H), 3.85 (s, 3H), 6.91-6.89 (d, J=8.2 Hz, 1H), 7.20-
7.17 (dd, J=8.2 Hz, 2.1Hz), 7.46 (d, J=2.0 Hz), 12.51 (s, 1H). 13
C
NMR (101 MHz, DMSO) δ ppm: 171.51, 168.42, 162.05,
157.49, 156.17, 150.02, 149.20, 148.19, 148.13, 121.51, 120.87,
120.68, 119.88, 115.99, 115.95, 109.94, 109.52, 55.80, 55.71,
34.91, 30.99, 28.23, 24.64, 22.01, 13.96. Molecular Formula:
C16H21N3O3S. Anal. Calcd: C (57.29), H (6.31), N (12.53), O
(14.31), S (9.56). Found: C (57.52), H (6.27), N (12.31), O
(13.87), S (10.03).
5.1.2.8.
N-[5-(4-hydroxy-3-methoxyphenyl)-1,
3,
4-
thiadiazol-2-yl)] nonamide (8). Yield: 57%; mp: 142°C; TLC: Rf
= 0.28 (60% ethyl acetate/ether petroleum). IR (KBr) ν cm-1:
1
3538.20, 3398, 3155.60, 2920.21-2730.72, 1680.21, 1573.98. H
NMR (400 MHz, CDCl3) δ ppm: 0.86-0.83 ppm (t, J= 6.8
Hz,3H), 1.50-1.24 (m, 10H), 1.86-1.79 (m, 2H), 2.78-2.76 (t, 7.7
Hz, 2H), 3.96 ( s, 3H), 5.9 (s, OH), 7.01-6.99 (d, J= 8.2 Hz, 1H),
7.43-7.41 (dd, J=8.2 Hz, 1.9 Hz, 1H), 7.52 (d, J= 1.7 Hz, 1H),
12.56 ( s, 1H). 13C NMR (101 MHz, CDCl3) δ ppm: 172.45,
163.40, 159.93, 148.56, 147.39, 123.24, 121.78, 115.40, 109.43,
56.52, 36.93, 32.26, 29.80, 29.72, 29.63, 25.93, 23.11, 14.52.
Molecular Formula: C18H25N3O3S. Anal. Calcd: C (59.48), H
(6.93), N (11.56), O (13.21), S (8.82). Found: C (59.55), H
(7.04), N (10.96), O (13.92), S (8.53).
5.1.2.13. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] heptanamide (14). Yield: 29.55%; mp: 192°C;
TLC: Rf = 0.27 (60% ethyl acetate/ether petroleum). IR (KBr) ν
1
cm-1: 3400.52, 3400.52, 3163.52, 2929.09-2863.98, 1696.07. H
NMR (400 MHz, DMSO) δ ppm: 0.86-0.82 (t, J=6.87Hz, 3H),
1.29-1.23 (m, 6H), 1.63-1.55 (m, 2H), 2.49-2.45 (t,
J=8.06Hz),3.33 (s, 3H), 7.46 (s, 1H), 7.76 (s, 1H), 10.01 (s, 1H),
12.55 (s, 1H). 13C NMR (101 MHz, DMSO) δ ppm: 171.56,
160.48, 157.91, 148.60, 147.34, 128.73, 123.32, 109.93, 84.89,
56.31, 56.22, 34.89, 30.95, 28.19, 24.60, 21.97, 13.95. Molecular
Formula: C16H21IN3O3S. Anal. Calcd: C (41.66), H (4.37), N
(9.11), O (10.40), S (6.95), I (27.51). Found: C (41.88), H (4.23),
N (8.99), O (11.70), S (5.17), I (28.03).
5.1.2.9. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] nonamide (12). Yield: 22.85%; mp: 123°C;
TLC: Rf = 0.31 (60% ethyl acetate/ether petroleum). IR (KBr) ν
1
cm-1: 3536.22, 3334.22, 3161.33, 2924.58-2853.71, 1694.22. H
NMR (400 MHz, CDCl3) δ ppm: 0.88-0.82 (dd , J= 14.1 Hz, 8,0
Hz,3H), 1.46-1.24 (m, 10H), 1.83-1.80 (m, 2H), 2.78-2.74 (t, 7.0
Hz, 2H), 3.98 (s, 3H), 7.44 (s, 1H), 7.85 (s, 1H), 9.77 (s, 1H),
12.74 (s, 1H).13C NMR (101 MHz, CDCl3) δ ppm: 172.47,
161.75, 160.29, 148.51, 146.70, 130.36, 124.71, 109.44, 56.91,
36.91, 32.26, 29.78, 29.71, 29.62, 25.86, 23.11, 14.53. Molecular
Formula: C18H24IN3O3S. Anal. Calcd: C (44.18), H (4.94), N
(8.59), O (9.81), S (6.55), I (25.93). Found: C (44.21), H (4.56),
N (8.37), O (8.18), S (5.33), I (29.35).
5.1.2.14. N-[5-(4-hydroxy-3-methoxyphenyl)-1,
3,
4-
thiadiazol-2-yl)] hexanamide (11). Yield: 41.11%; mp: 234°C;
TLC: Rf = 0.24 (60% ethyl acetate/ether petroleum). IR (KBr) ν
cm-1: 3402.67, 3402.67, 3162.49, 2997.19-2742.07, 1679.61. H
1
NMR (400 MHz, DMSO) δ ppm: 0.89-0.85 (t, J=6.8 Hz, 3H),
1.35-1.23 (m, 6H), 1.65-1.58 (m, 2H), (t, J=, 2H), 2.49-2.46 (t,
J=7.5 Hz, 2H), 3.86 (s, 3H), 6.89-6.87 (d, J= 8.2Hz, 1H), 7.33-
7.30 (dd, J= 8.2 Hz, 1.9Hz, 1H), 7.47-7.46 (d, J= 1.8Hz), 9.68 (s,
1H), 12.52 (s, 1H). 13C NMR (101 MHz, DMSO) δ ppm: 171.80,
170.06, 159.61, 159.03, 151.67, 141.90, 130.46, 127.92, 110.92,
93.61, 56.50, 34.65, 33.03, 26.55, 21.69, 13.67. Molecular
Formula: C15H19N3O3S. Anal. Calcd: C (56.06), H (5.96), N
(13.07), O (14.93), S (9.98). Found: C (56.39), H (6.04), N
(13.02), O (15.39), S (9.16).
5.1.2.10.
N-[5-(4-hydroxy-3-methoxyphenyl)-1,
3,
4-
thiadiazol-2-yl)] octanamide (9). Yield: 55.27%; mp: 139°C;
TLC: Rf = 0.27 (60% ethyl acetate/ether petroleum). IR (KBr) ν
1
cm-1: 3308. 01, 3154.41, 3154.41, 2946.86-2861.81, 1672.79. H
NMR (400 MHz, DMSO) δ ppm: 0.85-0.81 (t, J=6.9 Hz, 3H),
1.25-1.21 ( m, 8H), 1.61-1.55 (m, 2H), 2.47-2.44 (t, J= 6.8Hz,
2H), 3.83 (s, 3H), 6.9-6.87 (m, 1H), 7.30-7.28 (dd, J= 8.2 Hz, 2.1
Hz, 1H), 7.44 (d, J= 2.0 Hz, 1H), 9.71 (s, 1H), 12.50 (s, 1H).13C
NMR (101 MHz, DMSO) δ ppm: 171.49, 162.00, 157.46,
149.17, 148.10, 121.48, 120.64, 115.95, 109.92, 55.68, 34.87,
31.13, 28.49, 28.39, 24.65, 22.08, 13.96. Molecular Formula:
C17H23N3O3S. Anal. Calcd: C (58.43), H (6.63), N (12.02), O
(13.74), S (9.18). Found: C (58.45), H (5.97), N (14.64), O
(11.70), S (8.96).
5.1.2.15. N-[5-(4-hydroxy-3-iodo-5-methoxyphenyl)-1, 3, 4-
thiadiazol-2-yl)] hexanamide (15). Yield: 22.66%; mp: 205°C;
TLC: Rf = 0.24 (60% ethyl acetate/ether petroleum). IR (KBr) ν
cm-1: 3404.63, 3404.63, 3164.74, 2927.67-2735.99, 1699.04. H
1
NMR (400 MHz, DMSO) δ ppm: 0.88-0.85 (t, J= 6.8Hz, 3H),
1.32-1.25 (m, 4H), 1.65-1.58 (m, 2H), 2.49-2.46 (t, J=7.4Hz,
2H), 3.91 (s, 3H), 7.48-7.47 (d, J= 1.7 Hz, 1H), 7.78-7.77 (d, J=
1.8 Hz, 1H), 10.02 (s, 1H), 12.57 (s, 1H). 13C NMR (101 MHz,
DMSO) δ ppm: 171.54, 160.47, 157.91, 148.60, 147.91, 147.34,

ACCEPTED MANUSCRIPT
147.26, 128.74, 128.30, 124.12, 123.34, 109.91, 109.20, 84.87,
ANOVA test. Statistical significance was defined as P < 0.05.
Analysis was calculated using GraphPad Prism 5 software
(GraphPad software, California, USA).
84.66, 56.31, 56.23, 34.86, 30.74, 24.34, 21.86, 13.85. Molecular
Formula: C15H18IN3O3S. Anal. Calcd: C (40.28), H (4.06), N
(9.39), O (10.73), S (7.17), I (28.37). Found: C (40.61), H (3.98),
N (7.89), O (11.83), S (6.16), I (29.53).
5.3. Computational Methods
5.3.1. Docking
5.2 Biological Evaluation
Docking computations were carried out using Arguslab 4.0.1
(Planaria Software LLC, Seattle, WA) with AScore scoring
functions to find the lowest energy binding modes. The flexibility
of ligands was accounted for calculates of docking. The rat
TRPV1 model was used and the vanilloid binding site was
defined around of Tyr 510, Met 547, Trp 549 and Thr 550,
obtaining a box size of 14.51 x 10 x 24 A°. The docking
experiments were carried out with the following parameters:
Doking Engine: ArgusDock, Calculation Type: Dock and
Number poses: 150.
5.2.1. Compound preparation and delivery
The compounds were prepared as a stock solution of 10.000X
in DMSO and after for assay were diluted to a concentration of 1
µM in 99 % of Ringer buffer (140 mM NaCl, 5 mM KCl, 2
mM CaCl2, 1 mM MgCl2, 10 mM Glucose, 15 mM de HEPES
7.35 pH, 292 mOSM). Solutions of capsaicin (Sigma), BCTC
(ENZO life Science), Lanthanum chloride (Sigma) and
Ruthenium red (Alexis biochemicals) were used in a final
concentration of 30 nM, 1 µM, 100 mM and 10 µM, respectively.
5.2.2. HighThroughput calcium imaging assay: TRPV1
activation assay by capsaicin
5.3.2. Electrostatic Potential
The electrostatic potential were calculated using Arguslab
4.0.1 (Planaria Software LLC, Seattle, WA). First the energy of
docked poses was calculated by semi-empirical PM3 method at
RHF level of theory. The run type was set at SCF level with
maximum 200 iterations with a convergence of 10-10 Kcal/mol.
After the grids generated were used for obtain a map the
electrostatic potential onto a surface of the electron density.
Fluorescence measurements were carried out in real time PCR
thermocycler (StepOne plus, Applied Biosystem, USA), setted in
relative-quantification mode. The measurements were carried out
at 25°C in 13 cycles and registered each 10 seconds, using a filter
for fluoresceine (483-533 nM). Fluorescence recordings were
exported processed for analysis. Each measurement was
background subtracted considering non-capsaicin incubated
HEK-293T as base line. The relative fluorescence (F-F0/F0) was
calculated for every curve. The response was then normalized
with respect to the response of capsaicin-incubated cells. Data is
expressed as mean value ± standard deviation, from two
independent experiments (N=10).
6. Acknowledgements
We thank Dr. AM Zárraga Olavarría for her technical support.
The cTRPV1 clone was kindly provided by Dr. David Julius and
the rat TRPV1 model was kindly provided by Dr. Narender R.
Gavva. This work was supported by DIUC 210.023.046-1.0
(University of Concepcion) and FONDECYT grant 1110906
(SB). Brauchi Laboratory is part of CISNe-UACh.
5.2.3. Highthrouput calcium imaging assay: TRPV1
activation assay by temperature
Fluorescence measurements were carried out in real time PCR
thermocycler (StepOne plus, Applied Biosystem, USA), setted in
melting-curve mode. The temperature ramp (1°C / 35 seconds)
from 22°C up to 56°C. Controls for background were taken at
25°C. The measurements were carried out using a filter for
fluoresceine (483ex-533em). Fluorescence recordings were
exported processed for analysis. Each measurement was
background subtracted considering the signal from non-incubated
HEK-293T at 25°C as base line. The relative fluorescence (F-
F0/F0) was calculated for every curve. The response was then
normalized with respect to the response obtained for transfected
cells exposed at 50 °C of temperature. Normalized response to
temperature was expressed as mean ± standard deviation, from
two independent experiments run (N=3).
7. Abbreviations
DRG: dorsal root ganglion; rTRPV1: rat TRPV-1; cTRPV1:
chicken TRPV1; TLC: thin layer chromatography; Rf: retention
factor.
8. Associated content
Supporting Information. Temperature ramp for capsaicin on
rTRPV1, details for cell culture, transfection methods and
docking molecular for 1, 3, 4-thiadiazole derivatives.
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Highlights
Several 1, 3, 4-thiadiazole alkylamides have been synthetized as conformationally restricted
capsaicin analogues.
All compounds were evaluated on rat and chicken TRPV1 receptor.
1, 3, 4-thiadiazole derivatives showed to act as potent antagonist of TRPV1 receptor for
capsaicin and temperature activation mode.

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Supporting Information
Design and synthesis of conformationally restricted capsaicin analogues
based in the 1, 3, 4-thiadiazole heterocycle reveal a novel family of
transient receptor potential vanilloid 1 (TRPV1) antagonists
Carolyne Lespay Rebolledoa, Pamela Sotelo Hitshfeldb, Sebastián Brauchib and Miguel Zárraga O.a,
aDepartment of Organic Chemistry, Faculty of Chemical Sciences, University of Concepcion, Edmundo Larenas
160C, Concepción, 1430000, Chile.
bInstitute of Physiology, Faculty of Medicine, University Austral Of Chile, Campus Isla Teja 511-0566,Valdivia,
5090000, Chile.
* Corresponding author. Tel.: +56-41-2203921; fax: +56-41-2245974; e-mail: mzarraga@udec.cl
Table of contents
Figure 1. Temperature activation assay for capsaicin
Biological procedures
S2
S2
Docking Molecular into TRPV1 receptor
Spectra of Compounds 8, 11 and 12
S3-S8
S9-S14
Corresponding author. Tel.: +56-41-2203921; fax: +56-41-2245974; e-mail: mzarraga@udec.cl
S1

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Figure 1. Temperature-induced activation on rat TRPV1 receptors in transfected HEK-293T cells. The temperature
increment response curve is obtained from cells incubated in presence and absence (rTRPV1) of 30 nM of
capsaicin. Relative fluorescence normalised represent the fluorescence mean ± s. d. (n=3). Data obtained for the
agonist effect of capsaicin are statistically different with respect to rTRPV1 ***P < 0.05 (One-way ANOVA test).
Biological procedures
Cell Culture.
HEK-293T cells was cultured in a DMEM medium, supplemented with 5% of fetal bovin serum (HyClone), 50
U/ml penicillin (HyClone), 50mg/ml streptomycin (HyClone) and 2mM L-glutamin (GIBCO). The cultures were
supporting to 37°C temperature, in an ambient of 5% CO2 and 80% de relative humidity.
DNA Transfection
HEK-293T cells were transfected with pcDNA3 TRPV-1 of rat using Lipofectamine 2000 reagent (Invitrogen). In a
tube was added 97 µL Optimem (Gibco) and 3 µL de Lipofectamine 2000, in a second tube was added 1 µg of DNA
and a volume of Optimen until complete 100 µL. Both were incubate for 10 minutes. After the content of both tubes
was mixed and incubated for another additional 15 minutes additional. The mixture was added to RT-PCR plaques
of 60 mm with a confluence of 60% of cells and work with them 48hrs post transfection. Furthermore, HEK-293T
cells were transfected with pcDNA3 TRPV1 of chicken using Mirus Transfection Reagent (Mirus) according with
manufacture instructions. The mixture was added to plaques of 60 mm with a confluence of 60% of cells and work
with them 48 hrs post transfection.
Loading cells with Fluo-4AM
S2

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After 48 hours of transfection, cells were treated with 200 µL of trypsin, it incubates for 5 minutes at room
temperature, and then cells were soft pipetted and to transferred to a falcon tube of 15 mL and then were
centrifuged to a speed of at 6000 rpm for 10 minutes. After this cells were resuspended in 1 ml Ringer buffer (140
mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Glucose, 15 mM de HEPES 7.35 pH, 292 mOSM) , and
then 1 µl Fluo-4AM (Invitrogen) 1000X was added and incubated for 30 minutes at 37ºC. Later cells were
centrifuged and washed 3 times with PBS ringer buffer 1X, pH 7.34 for remove excess of fluorescent probe. Finally
cell pellet was resuspended in 1.5 ml Ringer buffer.
Docking Molecular into TRPV1 receptor
a) docked pose for capsaicin
b) docked pose for 5-iodononivamide
S3

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c) docked pose for BCTC
d) docked pose for 1, 3, 4-thiadiazole derivative 8
S4

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e) docked pose for 1, 3, 4-thiadiazole derivative 9
f) docked pose for 1, 3, 4-thiadiazole derivative 10
S5

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g) docked pose for 1, 3, 4-thiadiazole derivative 11
h) docked pose for 1, 3, 4-thiadiazole derivative 12
S6

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i) docked pose for 1, 3, 4-thiadiazole derivative 13
j) docked pose for 1, 3, 4-thiadiazole derivative 14
S7

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k) docked pose for 1, 3, 4-thiadiazole derivative 15
H1-NMR Spectrum of compound 8
S8

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C13-NMR spectrum of compound 8
S9

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H1-NMR Spectrum of compound 12
S10

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C13-NMR Spectrum of compound 12
S11

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H1-NMR Spectrum of compound 11
S12

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C13-NMR Spectrum of compound 11
S13

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S14

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