Tetrahydro-β-carboline derivatives targeting fatty acid amide hydrolase (FAAH) and transient receptor potential (TRP) channels

Article abstract of DOI:10.1016/j.bmcl.2012.10.137

A series of twenty-five derivatives of tetrahydro-β-carbolines 1-3 was synthesized and assayed on FAAH and TRPV1 and TRPA1 channels. Four carbamates, that is, 5a,c,e, and 9b inhibited FAAH with significant potency and interacted also effectively with TRPV1 and TRPA1 nociceptive receptors, while ureas 7b,d,f, and 8a,b were endowed with specific submicromolar TRPV1 modulating activities.

Full text of DOI:10.1016/j.bmcl.2012.10.137

Bioorganic & Medicinal Chemistry Letters 23 (2013) 138–142
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrahydro-b-carboline derivatives targeting fatty acid amide hydrolase
(FAAH) and transient receptor potential (TRP) channels
Giorgio Ortar a, , Luciano De Petrocellis b, Aniello Schiano Moriello b,c, Marco Allarà c, Enrico Morera a,
⇑
Marianna Nalli a, Vincenzo Di Marzo c
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy
b Endocannabinoid Research Group, Istituto di Cibernetica, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
c Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of twenty-five derivatives of tetrahydro-b-carbolines 1–3 was synthesized and assayed on FAAH
and TRPV1 and TRPA1 channels. Four carbamates, that is, 5a,c,e, and 9b inhibited FAAH with significant
potency and interacted also effectively with TRPV1 and TRPA1 nociceptive receptors, while ureas 7b,d,f,
and 8a,b were endowed with specific submicromolar TRPV1 modulating activities.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 October 2012
Revised 26 October 2012
Accepted 30 October 2012
Available online 12 November 2012
Keywords:
FAAH
Transient receptor potential channels
TRPV1
TRPA1
Tetrahydro-b-carbolines
The transient receptor potential vanilloid type 1 (TRPV1) is the
founding member of a large family of transient receptor potential
(TRP) channels, which typically acts as a molecular detector of nox-
ious signals in primary sensory neurons.1 The therapeutic potential
of targeting TRPV1 by agonists and antagonists, in particular for
chronic pain relief, has attracted an enormous amount of attention
over the past ten years.2 A wide range of stimuli are responsible for
TRPV1 activation including noxious heat, low extracellular pH and
a variety of chemical mediators. The prototypical endocannabinoid
anandamide (AEA) has been the first endogenous modulator of
TRPV1 to be identified, followed by other fatty acid conjugates of
biogenic amines such as N-arachidonoyldopamine, N-oleoyletha-
nolamine, and N-arachidonoylserotonin (AA-5-HT).3 In particular,
AA-5-HT exhibits interesting analgesic properties in models of
both inflammatory and neuropathic pain,4 owing to its peculiar
profile of ‘hybrid’ TRPV1 channel blocker and inhibitor of the endo-
cannabinoid degradative enzyme, fatty acid amide hydrolase
(FAAH), another attractive target involved in nociception.5 FAAH
inhibitors have in fact definitely demonstrated therapeutic benefit
in a variety of pain models.5
as conformationally restricted tryptamine derivatives, thus bearing
a certain resemblance with AA-5-HT.
More recently, TRPV1 affinity has been also demonstrated for
certain acyl amides of salsolinol (e.g., N-arachidonoylsalsolinol,
Fig. 1), an endogenous tetrahydroisoquinoline formed by a Pic-
tet–Spengler condensation of dopamine with acetaldehyde.8
Tetrahydro-b-carbolines such as 6-hydroxy-1,2,3,4-tetrahydro-
b-carboline
(1),9
6-hydroxy-1-methyl-1,2,3,4-tetrahydro-b-
carboline (2),10 and 1,2,3,4-tetrahydro-b-carboline (3),11 occurring
in mammals as a result of a reaction of serotonin or tryptamine
with formaldehyde or acetaldehyde,10,12 represent three con-
strained serotonin or tryptamine analogues. Therefore, we decided,
as a continuation of our previous studies on AA-5-HT,4 its ana-
logues,13 and other FAAH/TRP dual ligands based on the piperazi-
nylcarbamate and urea chemotypes,14 to prepare a number of
derivatives of carbolines 1–3 (compounds 4–9), and thus evaluate
the influence of the rigidification of the aminoethyl side chain of
the serotonin moiety of AA-5-HT on TRPV1 and FAAH activities
(Table 1). All compounds were tested on transient receptor poten-
tial ankyrin type-1 (TRPA1) as well.15 TRPA1 channel is expressed
in the polymodal C- and A-d fiber sensory neurons of the dorsal
root and trigeminal ganglia, is coexpressed with TRPV1 in a subset
of TRPV1-containing neurons, and is activated by noxious cold and
a variety of natural nocifensive or producing burning sensations
compounds, thus playing an important role in pain sensing.16 Since
all TRP channels are also rapidly desensitized by their agonists
Two natural indoloquinazolone alkaloids endowed with vanil-
loid activity, evodiamine6 and rutaecarpine7 (Fig. 1), can be viewed
⇑
Corresponding author. Tel.: +39 6 49913612; fax: +39 6 49913133.
E-mail address: giorgio.ortar@uniroma1.it (G. Ortar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.137

G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 138–142
139
HO
4
1
3
N
O
N
H
150
HN
N
H
O
O
9b
N-Arachidonoylserotonin
100
50
0
O
O
N
N
N
N
H
N
N
H
H
Me
Evodiamine
Rutaecarpine
R1
C-1 H2
C-3 H2
C-4 H2
aromatics
NH
HO
HO
8.0
7.0
6.0
5.0
4.0
3.0
ppm (t1)
N
Figure 2. 1H NMR spectrum of compound 9b in DMSO-d6 at room temperature.
NH
O
N
H
R2
N-Arachidonoylsalsolinol
Most of compounds 4–9 displayed complex 1H and 13C NMR
spectra at room temperature, indicating their existence as inter-
converting rotamers. As an example, the 1H NMR spectrum of com-
pound 9b is shown in Figure 2.
: R1 = OH; R2 = H
1
2: R1 = OH; R2 = Me
3: R1 = R2 = H
Figure 1. Structures of N-arachidonoylserotonin, alkaloids evodiamine and rutae-
carpine, N-arachidonoylsalsolinol, and tetrahydro-b-carbolines 1–3 (tryptamine
and phenethylamine moieties are highlighted in red).
In FAAH assays, amides 4 and ureas 7, 8, 9c,d were all essen-
tially inactive, whereas some of the carbamates (compounds 5a–
c,e and 9b) exhibited significant FAAH inhibitory activities. Amides
4, with the partial exception of 4d,e, had also modest effects on
TRPV1 and TRPA1 channels. Carbamates with meta-substituted
aromatic rings were more potent on FAAH than the para-substi-
tuted ones (compare compounds 5a,e and 5d,f), in line with previ-
ous results on AA-5-HT analogues,13 piperazinyl carbamates and
ureas,14 and arylcarbamic acid aryl esters.19 The presence of a
methyl group on the tetrahydro-b-carboline moiety was detrimen-
tal to the inhibitory potency (compare compounds 5e and 6). In
contrast, removal of the 6-OH group from carbamate 5e resulted
in a ꢀsix fold increase in potency (compound 9b), a result opposite
to that previously described by Fowler et al. for N-arachidonoyl-
tryptamine, a 2.3-fold weaker inhibitor of AEA hydrolysis than
AA-5-HT.20 Most of the carbamates were able to modulate both
HO
HO
a
NH
N
R
N
H
N
H
O
1
4
Scheme 1. Synthesis of compounds 4. Reagents and conditions: (a) RCO2H, HOBt/
EDC, DMF, rt, 1 h, then 1, Et3N, DMF, rt, 16 h.
after activation,17 we also tested the capability of the compounds
to inhibit the activation of TRPV1 or TRPA1 by their canonical ago-
nists, capsaicin and allyl isothiocyanate. Therefore, for each com-
pound, both EC50 (for activation) and IC50 (for desensitization)
values were calculated. However, when exhibiting an EC50 >10
and an IC50 in the low M range, a compound is likely to behave as
a ‘true’ antagonist rather than as a desensitizer.
The synthesis of amides 4 was carried out by condensation of 1
(as the hydrochloride) with the appropriate carboxylic acids using
1-hydroxybenzotriazole (HOBt)/N-ethyl-N0-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDC) as the carboxylate activator
(Scheme 1). Carbamates 5, 6, 9a,b and ureas 7, 8, 9c,d were synthe-
sized by condensation of b-carbolines 1 (as the hydrochloride), 2,
and 3 (as the hydrochloride) with the appropriate aryl chlorofor-
mates or isocyanates (Scheme 2).18
TRPV1 and TRPA1 channels with EC50 and/or IC50 values <10 lM
(compounds 5a,c–f, and 9a,b). Four carbamates, that is 5a,c,e,
and 9b, acted as efficient triple FAAH/TRPV1/TRPA1 ligands and
could conceivably represent useful leads for novel analgesic com-
pounds targeting FAAH and additional players in pain. Carbamate
5b was the only dual FAAH/TRPV1 blocker identified, but with
potencies lower than those of AA-5-HT.
With the exception of 7c, all ureas showed good and selective
TRPV1 modulating activities. Particularly interesting in this respect
appeared to be compound 7b, endowed with selective activity in
the low nanomolar range. Recent investigations have been devoted
to the identification of TRPV1 regions involved in the recognition of
some ligands and in the prediction of their binding modes.21 The
lM
l
R1
R1
R1
a
or
NH
N
OR3
N
NHR3
N
H
N
H
N
H
R2
R2
O
R2
O
1
1
1
: R = OH; R2 = H
: R = OH; R2 = H
: R = OH; R2 = H
1
5
7
2: R1 = OH; R2 = Me
3: R1 = R2 = H
6: R1 = OH; R2 = Me
9a,b: R1 = R2 = H
8: R1 = OH; R2 = Me
9c,d: R1 = R2 = H
Scheme 2. Synthesis of compounds 5–9. Reagents and conditions: (a) R3OCOCl or R3NCO, Et3N, DMF, rt, 16 h.

140
G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 138–142
presence of a 4-t-Bu phenyl moiety in the lipophilic arm of TRPV1
modulators that can be traced back to capsaicinoids and resinifera-
toxin (RTX) was found frequently to be critical for potency, pre-
efficacy TRPA1 agonist activity and ‘true’23 TRPV1 antagonist prop-
erties, respectively, and deserve a special mention.
The EC50 and IC50 values at the same TRP channel were gener-
ally of the same order of magnitude and thus, in most cases, acti-
vation and desensitization potencies were comparable; some
discordances were, however, also evident, particularly for the
TRPA1 channel (see compounds 4a–c, 7b,f), suggesting in these
sumably related to optimal
p–p stacking and hydrophobic
interactions.22 1-Methyl group on the carboline moiety reduced
the activity of ureas for TRPV1 (compare compounds 7b,d and
8a,b).
The influence of the removal of the 6-OH substituent on the TRP
activities was not uniform and both a reduction (compare com-
pounds 5d, 7b and 9a,c) and an improvement (compare com-
pounds 5e and 9b) of potencies, but not necessarily of efficacy,
was observed. Therefore, no definite conclusion can be drawn as
to the role of the 6-OH group in TRPV1 and TRPA1 modulation.
Anyway, among carbamates and ureas devoid of the 6-OH substi-
tuent, 9b and 9c exhibited submicromolar FAAH inhibitory/low
cases either the occurrence of antagonism (when the IC50 <<EC50)
or a poor capability of producing desensitization (when the
EC50 <<IC50).
Comparison of AA-5-HT and its analogues13 and tetrahydro-b-
carboline derivatives with identical R (or R3) groups revealed that
rigidification of the serotonin side chain generally reduced the
activity for FAAH and TRPV1, with only few exceptions (i.e., com-
pounds 5a,d for TRPV1).
Table 1
Results of FAAH, TRPV1, and TRPA1 assays of derivatives of tetrahydro-b-carbolines 1,2, and 3a
HO
R1
R1
N
R
N
OR3
N
NHR3
NH
NH
NH
O
R2
O
R2
O
4
7: R1 = OH; R2 =H
5: R1 = OH; R2 = H
: R1 = OH; R2 = Me
: R1 = OH; R2 = Me
9a b
8
6
: R1 = R2 = H
9c d
,
: R1 = R2 = H
,
Compound R or R3
FAAH (IC50
,
TRPV1b
(efficacy)
TRPV1 (EC50
,
TRPV1c (IC50
,
TRPA1d
TRPA1 (EC50
M)
,
TRPA1e (IC50
M)
,
lM)
lM)
lM)
(efficacy)
60.2 0.1
l
l
4a
>10
<10
ND
9.30 0.90
5.24 0.01
35.49 0.77
4b
4c
>10
>10
<10
<10
ND
ND
10.60 0.30
9.60 0.03
< 10
ND
ND
11.67 0.81
20.17 0.35
16.7 0.1
4d
4e
4f
5a
5b
5c
5d
5e
5f
Ph-4-t-Bu
Ph-4-Ph
>10
>50
>50
3.69 0.84
6.77 0.85
6.25 0.82
>10
1.74 0.03
>10
>10
>10
>10
>10
>10
>50
>50
>50
71.8 0.9
49.0 1.6
48.6 0.5
52.3 0.5
<10
0.54 0.04
6.42 0.54
11.80 0.39
5.1 2.3
ND
0.44 0.01
3.13 0.35
19.45 0.30
6.20 0.10
6.40 0.10
5.10 0.10
0.55 0.5
9.60 0.20
11.38 0.37
ND
64.9 0.8
83.5 2.0
109.6 2.9
65.4 0.9
113.0 0.8
81.4 2.6
73.9 4.6
77.5 0.4
73.4 0.1
59.1 0.1
38.1 0.8
16.51 0.59
4.09 0.56
7.78 0.86
4.95 0.10
14.30 0.25
1.68 0.32
4.29 1.03
1.70 0.04
5.04 0.03
1.71 0.02
17.04 1.09
ND
19.57 0.75
21.03 0.10
14.76 0.86
1.82 0.75
10.77 0.10
13.77 0.62
9.81 0.83
0.20 0.04
1.92 0.29
3.71 0.25
30.60 0.83
12.90 0.65
15.30 0.60
10.47 0.84
26.32 0.46
3.25 0.36
3.40 0.51
3.48 0.17
9.76 0.30
5.36 0.20
34.87 0.12
21.11 0.57
55.36 2.72
24.56 0.15
17.03 0.36
18.84 0.67
22.50 0.68
35.12 2.85
3.36 0.38
2.84 0.43
3.90 0.18
7.14 0.27
CH2Ph-3-Ph
Ph-3-t-Bu
Ph-3-CF3
Ph-3-(CH2)4CH3
Ph-4-t-Bu
Ph-3-Ph
<10
ND
54.4 0.4
12.5 0.1
37.6 0.8
< 10
0.4 0.01
5.2 0.1
7.75 0.57
ND
Ph-4-Ph
Ph-3-Ph
6
7a
7b
7c
7d
7e
7f
8a
8b
9a
9b
9c
9d
Ph-3-t-Bu
Ph-4-t-Bu
Ph-3-CF3
Ph-4-CF3
Ph-3-Ph
30.7 0.2
69.2 0.7
15.5 1.3
57.3 0.2
78.3 1.6
73.9 2.6
68.2 0.1
67.4 0.3
17.30 0.01
20.6 1.8
<10
1.89 0.07
5.35 0.42
0.019 0.0012 0.001 0.00001 17.9 0.1
7.43 0.50
0.26 0.07
2.27 0.20
0.17 0.03
1.2 0.02
0.47 0.01
5.29 0.01
2.40 0.94
ND
13.82 0.77
0.33 0.02
1.86 0.11
0.14 0.01
0.098 0.05
0.31 0.01
13.26 1.23
7.01 0.29
0.75 0.01
4.36 0.67
88.8 1.1
91.6 2.1
136.5 1.5
93.3 5.8
117.5 5.7
70.5 1.0
117.8 3.5
33.4 0.9
52.6 1.2
102.2 1.8
Ph-4-Ph
Ph-4-t-Bu
Ph-4-CF3
Ph-4-t-Bu
Ph-3-Ph
Ph-4-t-Bu
Ph-4-CF3
>50
>50
0.275 0.04
>50
>50
<10
ND
ND, not determined when efficacy is lower than 10%.
a
Data are means SEM of N = 3 determinations.
b
As percent of ionomycin (4
l
M).
Determined against the effect of capsaicin (0.1
As percent of allyl isothiocyanate (100 M).
Determined against the effect of allyl isothiocyanate (100
c
d
e
l
M).
l
lM).

G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 138–142
141
and 10 mM HEPES, pH 7.4), resuspended in the same buffer, and transferred
(about 100,000 cells) to the quartz cuvette of the spectrofluorimeter (Perkin-
Elmer LS50B equipped with PTP-1 Fluorescence Peltier System; PerkinElmer
Life and Analytical Sciences, Waltham, MA, USA) under continuous stirring. The
In conclusion, in this Letter we have presented a series of tetra-
hydro-b-carboline derivatives designed as rigid analogues of AA-5-
HT and its congeners and have identified: (1) some carbamates
that inhibit FAAH with significant potency and interact also effec-
tively with TRPV1 and TRPA1 nociceptive receptors, and (2) a num-
ber of ureas endowed with submicromolar TRPV1 modulating
activities.
changes in [Ca2+
] were determined before and after the addition of various
i
concentrations of test compounds by measuring cell fluorescence
(kEX = 488 nm, kEM = 516 nm) at 25 °C. Curve fitting (sigmoidal dose-response
variable slope) and parameter estimation were performed with GraphPad
PrismÒ (GraphPad Software Inc., San Diego, CA). Potency was expressed as the
concentration of test substances exerting a half-maximal agonist effect (i.e.,
half-maximal increases in [Ca2+]i) (EC50). In the case of TRPV1 assays, the
efficacy of the agonists was first determined by normalizing their effect to the
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with 100
was evaluated against AITC (100
l
M allyl isothiocyanate (AITC). Antagonist/desensitizing behaviour
l
M) for TRPA1 and capsaicin (0.1 M) for
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TRPV1, by adding the test compounds in the quartz cuvette 5 min before
stimulation of cells with agonists. Data are expressed as the concentration
exerting a half-maximal inhibition of agonist-induced [Ca2+]i elevation (IC50),
which was calculated again using GraphPad PrismÒ software. The effect on
[Ca2+]i exerted by agonist alone was taken as 100%. Dose response curves were
fitted by a sigmoidal regression with variable slope. All determinations were
performed at least in triplicate. Statistical analysis of the data was performed
by analysis of variance at each point using ANOVA followed by the Bonferroni’s
test.
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18. General procedure for the synthesis of compounds 4. To a stirred solution of the
appropriate carboxylic acid (0.20 mmol) in DMF (1 mL) HOBt (35 mg,
0.24 mmol) and EDC (40 mg, 0.24 mmol) were added at 0 °C. The mixture
was stirred for 15 min at 0 °C and for 1 h at room temperature. Then 1 (as
hydrochloride, 54 mg, 0.24 mmol) and Et3N (34 lL, 0.24 mmol) were added,
and the mixture was stirred overnight at room temperature. The mixture was
diluted with brine and extracted with AcOEt. The organic phase was washed
with 2 N HCl solution, saturated NaHCO3, and brine, dried (Na2SO4), and
evaporated under vacuum. The residue was purified by column
chromatography. General procedure for the synthesis of compounds 5, 6, 9a,b.
To a stirred 20% phosgene solution in toluene (0.60 mL, 1.15 mmol) a solution
of the appropriate phenol (0.29 mmol) and Et3N (47 lL, 0.34 mmol) in dry
toluene (2.9 mL) was added dropwise at 0 °C. The reaction mixture was stirred
for 3 h at room temperature and evaporated under vacuum. The residue of the
crude chloroformate was dissolved in dry CH2Cl2 (2 mL) and a solution of 1 (as
hydrochloride) or 2 or 3 (as hydrochloride) (0.29 mmol) and Et3N (in the case
of 1 and 3, 81 lL, 0.58 mmol; in the case of 2, 41 lL, 0.29 mmol) in dry DMF
(1 mL) was added dropwise at room temperature with stirring. The reaction
mixture was stirred overnight at room temperature, diluted with water, and
extracted with AcOEt. The organic phase was washed twice with brine, dried
(Na2SO4), and evaporated under vacuum. The residue was purified by column
chromatography. General procedure for the synthesis of compounds 7, 8, 9c,d. A
solution of the appropriate isocyanate (0.24 mmol), 1 (as hydrochloride) or 2 or
13. Ortar, G.; Cascio, M. G.; De Petrocellis, L.; Morera, E.; Rossi, F.; Schiano Moriello,
A.; Nalli, M.; de Novellis, V.; Woodward, D. F.; Maione, S.; Di Marzo, V. J. Med.
Chem. 2007, 50, 6554.
14. Morera, E.; De Petrocellis, L.; Morera, L.; Schiano Moriello, A.; Ligresti, A.; Nalli,
M.; Woodward, D. F.; Di Marzo, V.; Ortar, G. Bioorg. Med. Chem. Lett. 2009, 19,
6806.
15. FAAH assays. The effect of increasing concentrations of the new synthetic
compounds on the enzymatic hydrolysis of [14C]anandamide was studied by
using membranes prepared from rat brain. In brief, the whole rat brain was
homogenized at 4 °C in 50 mM Tris-HCl buffer, pH 7.0, by using an ultraturrax
and a dounce homogenizer. Homogenates were first centrifuged at 800 g to get
rid the debris and the supernatant was centrifuged at 10,000 g. The pellet from
3 (as hydrochloride) (0.24 mmol), and Et3N (in the case of 1 and 3, 67
lL,
0.48 mmol; in the case of 2, 34 L, 0.24 mmol) in dry DMF (1 mL) was stirred
l
overnight at room temperature. The mixture was diluted with brine and
extracted with AcOEt. The organic phase was washed twice with brine, dried
(Na2SO4), and evaporated under vacuum. The residue was purified by column
chromatography. Data for selected compounds: Compound 4d: yield 31%; mp
256 °C; IR (KBr) 3312, 3120, 2960, 1585, 1448, 1367, 1203 cmꢁ1 1H NMR
;
(300 MHz, DMSO-d6) d1.32 (9H, s), 2.51 and 2.69 (2H, 2 br s), 3.63 and 3.96 (2H,
2 br s), 4.59 and 4.77 (2H, 2 br s), 6.56–7.13 (3H, m), 7.40 (2H, d, J = 7.5 Hz),
7.49 (2H, d, J = 7.5 Hz), 8.66 (1H, s), 10.32 and 10.62 (1H, 2 br s); 13C NMR
(75 MHz, DMSO-d6) d 20.14, 21.68, 30.33, 30.92, 34.46, 45.49, 101.81, 110.66,
111.21, 124.80, 125.01, 125.11, 126.64, 127.17, 130.31, 131.07, 133.27, 150.35,
152.17. Compound 5e: yield 43%; oil; IR (CHCl3) 3468, 3359, 2926, 1708, 1600,
this latter centrifugation was used for the assay. Membranes (70–100
incubated with increasing concentrations (up to 50 M) of the test compounds
and [14C]AEA (10,000 cpm, 1.8
M) in 50 mM Tris–HCl, pH 9, for 30 min at
37 °C.
14C]Ethanolamine produced from 14C]AEA hydrolysis was used to
lg) were
l
l
[
[
1417, 1259, 1184, 1074 cmꢁ1 1H NMR (300 MHz, CD3OD) d 2.74 and 2.79 (2H,
;
calculate FAAH activity and was measured by scintillation counting of the
aqueous phase after extraction of the incubation mixture with 2 volumes of
CHCl3/CH3OH (1:1 by volume). Data are expressed as the concentration
exerting a half-maximal inhibition (IC50). TRPV1 and TRPA1 channel assays.
HEK293 (human embryonic kidney) cells stably over-expressing recombinant
rat TRPA1, or human TRPV1 were grown on 100 mm diameter Petri dishes as
mono-layers in minimum essential medium (EMEM) supplemented with non-
essential amino acids, 10% foetal bovine serum, and 2 mM glutamine, and
maintained at 5% CO2 at 37 °C. Stable expression of each channel was checked
by quantitative PCR (data not shown). The effect of the substances on
intracellular Ca2+ concentration ([Ca2+]i) was determined by using Fluo-4, a
selective intracellular fluorescent probe for Ca2+. On the day of the experiment,
cells were loaded for 1 h at room temperature with the methyl ester Fluo-4-AM
2 m), 3.84 and 3.94 (2H, 2 m), 4.68 and 4.83 (2H, 2 br s), 6.64–7.57 (12H, m);
13C NMR (75 MHz, CD3OD) d 21.93, 22.51, 43.59, 43.71, 44.28, 49.84, 103.25,
111.92, 111.94, 112.32, 121.40, 121.64, 123.18, 125.12, 127.86, 127.96, 128.29,
128.66, 128.84, 129.85, 130.71, 131.88, 141.30, 143.94, 151.32, 153.10, 153.13,
155.92, 155.95. Compound 7b: yield 70%; mp 158–161 °C; IR (KBr) 3328, 2960,
1636, 1593, 1518, 1417, 1239 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d 1.25 (9H,
;
s), 2.68 (2H, br s), 3.80 (2H, br s), 4.64 (2H, s), 6.57 (1H, d, J = 8.6 Hz), 6.74 (1H,
br s), 7.10 (1H, J = 8.6 Hz), 7.25 (2H, d, J = 8.7 Hz), 7.39 (2H, d, J = 8.7 Hz); 13C
NMR (75 MHz, DMSO-d6) d 21.14, 31.21, 33.76, 42.18, 42.39, 101.84, 106.17,
110.49, 111.16, 119.60, 119.71, 124.77, 127.33, 130.40, 132.12, 137.84, 144.01,
150.27, 155.54. Compound 9b: yield 49%; mp 156–159 °C; IR (KBr) 3384, 3046,
2845, 1698, 1624, 1433, 1416, 1226, 1180, 1077 cmꢁ1 1H NMR (300 MHz,
;
(4
EMEM without foetal bovine serum, then were washed twice in Tyrode’s buffer
(145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 10 mM -glucose,
lM in dimethyl sulfoxide containing 0.02% Pluronic F-127, Invitrogen) in
CDCl3) d 2.91 (2H, br s), 3.90 and 3.99 (2H, 2 br s), 4.74 and 4.84 (2H, 2 br s),
7.11–7.56 (13H, m), 8.37 and 8.56 (1H, 2 br s); 13C NMR (75 MHz, CDCl3) d
21.02, 21.60, 42.61, 43.11, 110.99, 111.06, 117.87, 118.08, 119.45, 119.59,
D

142
G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 138–142
120.55, 120.58, 121.72, 121.89, 124.27, 127.17, 127.64, 128.79, 129.66, 136.29,
140.20, 142.79, 151.74, 154.56.
19. Tarzia, G.; Duranti, A.; Tontini, A.; Piersanti, G.; Mor, M.; Rivara, S.; Plazzi, P. V.;
Park, C.; Kathuria, S.; Piomelli, D. J. Med. Chem. 2003, 46, 2352.
Lazar, J.; Pearce, L. V.; Pavlyukovets, V. A.; Blumberg, P. M.; Choi, S. J. Comput.
Aided Mol. Des. 2011, 25, 317; (c) Jara-Oseguera, A.; Nieto-Posadas, A.; Szallasi,
A.; Islas, L. D.; Rosenbaum, T. Open Pain J. 2010, 3, 68; (d) Kym, P. R.; Kort, M. E.;
Hutchins, C. W. Biochem. Pharmacol. 2009, 78, 211.
20. Fowler, C. J.; Tiger, G.; Lopez-Rodriguez, M. L.; Viso, A.; Ortega-Gutierrez, S.;
Ramos, J. A. J. Enzyme Inhib. Med. Chem. 2003, 18, 225.
22. Vriens, J.; Appendino, G.; Nilius, B. Mol. Pharmacol. 2009, 75, 1262.
23. Inhibition without agonism per se, and hence not due to desensitization.
21. (a) Wang, Z.; Sun, L.; Yu, H.; Zhang, Y.; Gong, W.; Jin, H.; Zhang, L.; Liang, H. Int.
J. Mol. Sci. 2012, 13, 8958; (b) Lee, J. H.; Lee, Y.; Ryu, H. C.; Kang, D. W.; Lee, J.;