Arylboronic acids as dual-action FAAH and TRPV1 ligands

Article abstract of DOI:10.1016/j.bmcl.2016.01.071

A series of 31 arylboronic acids designed on the basis of the pharmacophore model for a variety of TRPV1 antagonists was prepared and tested on FAAH and TRPV1 channel. Four of them, that is, compounds 3c, 4a, 5a,b acted as dual FAAH/TRPV1 blockers with IC₅₀ values between 0.56 and 8.11 μM whereas ten others (compounds 1c,f-i, 2c-f, 4b) inhibited FAAH and activated/desensitized TRPV1.

Full text of DOI:10.1016/j.bmcl.2016.01.071

Accepted Manuscript
Arylboronic acids as dual-action FAAH and TRPV1 ligands
Enrico Morera, Vincenzo Di Marzo, Ludovica Monti, Marco Allarà, Aniello
Schiano Moriello, Marianna Nalli, Giorgio Ortar, Luciano De Petrocellis
PII:
S0960-894X(16)30072-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.071
BMCL 23536
Reference:
Bioorganic & Medicinal Chemistry Letters
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22 January 2016
23 January 2016
Please cite this article as: Morera, E., Marzo, V.D., Monti, L., Allarà, M., Moriello, A.S., Nalli, M., Ortar, G.,
Petrocellis, L.D., Arylboronic acids as dual-action FAAH and TRPV1 ligands, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.071
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Bioorganic & Medicinal Chemistry Letters
Arylboronic acids as dual-action FAAH and TRPV1 ligands
Enrico Moreraa,*, Vincenzo Di Marzob, Ludovica Montia, Marco Allaràb, Aniello Schiano Moriellob,
Marianna Nallia, Giorgio Ortara, Luciano De Petrocellisb,*
aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy
bEndocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 31 arylboronic acids designed on the basis of the pharmacophore model for a variety
of TRPV1 antagonists was prepared and tested on FAAH and TRPV1 channel. Four of them,
that is, compounds 3c, 4a, 5a,b acted as dual FAAH/TRPV1 blockers with IC50 values between
0.56 and 8.11 M whereas ten others (compounds 1c,f-i, 2c-f, 4b) inhibited FAAH and
activated/desensitized TRPV1.
2009 Elsevier Ltd. All rights reserved.
Keywords:
Fatty acid amide hydrolase
FAAH
Transient receptor potential vanilloid type-1 channel
TRPV1
Dual-ligands
Boronic acids
In recent years, there has been an increasing interest in the
therapeutic potential of boron-containing compounds as enzyme
inhibitors, boron neutron capture therapy (BNCT) agents, and
drug delivery devices.1 For example, bortezomib, a dipeptidyl
boronic acid approved in 2003 by the FDA for the treatment of
multiple myeloma (MM) and mantle cell lymphoma, stands out
as one of the most potent proteasome inhibitors so far available.2
Boronic acids inhibit hydrolytic enzymes such as serine
proteases1e,3 through the formation of a tetracoordinate-boronate
complex by coordination of the catalytic serine residue, thus
acting as transition-state analogues.1e,4 In 2008, Minkkilä et al.
reported that a series of commercially available phenyl-,
heteroaryl-, alkyl-, and alkenylboronic acids behaved as potent
and selective fatty acid amide hydrolase (FAAH) inhibitors5 and,
at the same time, some boronic acid derivatives were patented as
reversible FAAH inhibitors by Infinity Pharmaceuticals.6
FAAH is a member of the amidase signature family of serine
hydrolases7 that cleaves and regulates a broad range of
endogenous signalling lipid amides, such as the prototypical
endocannabinoid anandamide (AEA),8 the anti-inflammatory
substance palmitoylethanolamide (PEA),9 the sleep-inducing
agent oleamide (OA),10 and the analgesic and satiety regulating
mediator oleoylethanolamide (OEA).11 Thus, blockade of FAAH
represents a promising approach for the treatment of various
disease states such as pain,12 inflammatory,13 and
neuropsychiatric disorders,14 and, toward this ends, many classes
of FAAH inhibitors, including -ketoheterocycles, carbamates,
and ureas, have been reported.15 Although in animal models
FAAH inhibitors have proved to be effective analgesic agents,
devoid of the CNS side effects that arise when a cannabinoid
receptors agonist is used,16 data on their clinical efficacy are less
encouraging. For example, the potent and selective FAAH
inhibitor PF-04457845, evaluated as an analgesic in randomized,
placebo-controlled phase II clinical trials, was apparently
ineffective.17 Among the reasons for this failure, the fact that
FAAH inhibition might indirectly lead to activation of other,
non-cannabinoid receptors involved in nociception, such as
transient receptor potential vanilloid 1 (TRPV1) channel, should
be taken into account.18 TRPV1, a non-selective cation channel
belonging to the large family of transient receptor potential
(TRP) ion channels,19 is activated by a variety of stimuli,
including noxious heat, protons, endogenous substances such as
AEA, OEA, and lipoxygenase products, and exogenous
compounds such as capsaicin (Figure 1), and has emerged as a
promising therapeutic target for pain management.
Recent findings of us suggest that compounds that inhibit both
FAAH and TRPV1 may be more efficacious in pain relief than
those targeting only one such protein.20 As continuation of our
efforts to identify new molecules able to target simultaneously
both FAAH and TRPV1 receptors, we hypothesized that the
incorporation of a boronic acid group (as the FAAH inhibiting
moiety) into the pharmacophore model for a variety of TRPV1
antagonists (Figures 1 and 2)19h,l could represent a simple strategy
for the development of combined FAAH/TRPV1 blockers. The
model can be generalized as a central hydrogen-bond acceptor/
______
* Corresponding authors. Tel.: +39 6 49913626; fax: +39 6 49913133 (E.M.);
tel.: +39 081 8675173 (L.D.P.).
E-mail addresses: enrico.morera@uniroma1.it (E. Morera),
luciano.depetrocellis@icb.cnr.it (L. De Petrocellis).

Scheme 1. Reagents and conditions: (a) R2CO2H, HOBt/EDC, DMF, rt, 1 h,
then appropriate amine, Et3N, DMF, rt, 16 h. (b) NaIO4, 2 N HCl, THF/H2O
(3:1), rt, 3-5 h.
Figure 1. Structure of capsaicin and of selected TRPV1 antagonists.
Scheme 2. Reagents and conditions: (a) PhNTf2, Et3N, CH2Cl2, rt, 16 h; (b)
B2pin2, PdCl2, DPPF, AcOK, dioxane, 100 °C, 4h; (c) TFA/CH2Cl2 (3:1), rt.,
40 min.
Figure 2. Design of arylboronic acid derivatives as FAAH/TRPV1 blockers.
donor motif flanked by a lipophilic tail on one side and an aromatic
group that incorporates a hydrogen-bond acceptor on the other
side.
Accordingly, 31 arylboronic acids inspired by this model were
prepared and tested on FAAH and TRPV1 channel (compounds
1-7, Table 1).
All the boronic acids were prepared as pinacol boronate esters
and subsequent deprotection with sodium metaperiodate.21 In
detail, amides 1, 3, and 7 were synthesized by acylation of the
corresponding aminobenzyl or anilino boronates 8 with the
appropriate carboxylic acids R2CO2H, using 1-hydroxybenzotriazole
(HOBt)/N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDC) as the carboxylate activator (Scheme 1), followed
by cleavage of the boronates 9.22 The non-commercially available
4-(aminomethyl)-2-methoxyphenylboronic acid pinacol ester was
prepared by a palladium-catalyzed cross-coupling reaction of
diboron pinacol ester (B2pin2) with the appropriate aryl triflate
(Scheme 2).23 Amides 2 and 4 were obtained by reaction of the
activated carboxylic acids 10 with the appropriate amines RNH2,
followed by cleavage of the boronates 11 (Scheme 3).22 Ureas 5
and 6 were prepared by reaction of the corresponding 4-
Scheme 3. Reagents and conditions: (a) HOBt/EDC, DMF, rt, 1 h, then
RNH2, DMF, rt, 16 h; (b) NaIO4, 2 N HCl, THF/H2O (3:1), rt, 3-5 h.
aminobenzyl or anilino boronates
8
with 4-tert-butyl-
phenylisocyanate followed by cleavage of the boronates 12.22
The effects of arylboronic acids 1-7 on [14C]anandamide
hydrolysis by rat membranes (which express FAAH as the only
AEA hydrolyzing enzyme) and on intracellular Ca2+ elevation in
HEK293 cells stably transfected with the human TRPV1 cDNA
are shown in Table 1.24 The TRPV1 antagonist or desensitizing
activity was assessed by adding the test compounds 5 min before
stimulation of cells with the reference agonist capsaicin. Data
obtained for the two most potent boronic acids reported by
Minkkilä et al.5 (compounds 13 and 14), compounds 15, 16,25 the
selective TRPV1 antagonist SB-366791,26 and two 'dual-target'
agents previously identified by us [N-arachidonoylserotonin
(AA-5-HT) and the piperazinyl carbamate OMDM-198]20 are
also included in the Table.
The majority of arylboronic acids 1-7 showed, as expected,
fairly good FAAH inhibitory activities (compounds 1c,e-i, 2c-f,
3c-e, 4a-c,e-g, 5a,b), irrespective of the nature of the functional
group X-Y connecting arylboronic acid and lipophilic moieties
and of the presence or absence of a methylene bridge. The boronic
acid functionality was essential for FAAH inhibitory activity, as
Scheme 4. Reagents and conditions: (a) tBu-4-PhNCO, Et3N, DMF, rt, 16 h;
(b) NaIO4, 2 N HCl, THF/H2O (3:1), rt, 3-5 h.
demonstrated by the lack of activity of the analogues of 2f, 15
and 16. The choice of 15 and 16 as reference compounds was
driven by the high, submicromolar modulating potency of the
boronic acid 2f on both FAAH and TRPV1. Notably, however,
the movement of the B(OH)2 group away from the para-position
resulted in a loss of activity (compare compounds 3c and 7). In
their study, Minkkilä et al.5 found that para-substituted
compounds were indeed more potent FAAH inhibitors than the
meta-substituted ones, a result which was ascribed to the more
steric tolerance near the enzyme's catalytic site for the para-
substitution. A similar explanation may be invoked for the
decrease in activity of the 2-methoxy-substituted compounds
1h,i, 5b as compared to their unsubstituted counterparts 1f,g, 5a.
The choice of an -arylaliphatic chain as the lipophilic tail
appeared to be beneficial for optimal activity on FAAH with a
positive relationship with the chain length (compare compounds
1d,f, and g; 2b,d,e, and f; 3d and e; 4c,f, and g). Conversely, the

Table 1. Results of TRPV1 and FAAH assays of arylboronic acids 1-7, 13, and 14a
FAAH
(IC50 M)
TRPV1
TRPV1
(EC50 μM)
TRPV1
Compound
R1
H
R or R2
(efficacy %)b
(IC50 μM)c
42.2
0.022
0.020
0.047
0.032
> 10
> 10
1a
OMe
49.2
1b
H
H
Ph-4-Ph
(CH2)2Ph
22.0
60.2
< 10
41.1
54.7
59.3
62.5
< 10
< 10
27.4
44.9
62.8
52.5
39.3
< 10
< 10
< 10
< 10
< 10
12.5
< 10
< 10
< 10
< 10
< 10
< 10
< 10
4.23
0.32
ND
7.11
0.23
> 10
2.89
0.50
1.53
0.034
> 10
> 10
5.53
2.19
2.53
0.37
0.78
> 10
1.39
> 10
> 10
0.75
> 10
> 10
> 10
> 10
> 10
> 10
4.13
8.11
0.33
> 10
1.51
1.08
0.47
1.32
1.77
> 10
> 10
0.28
1.17
0.63
0.25
> 10
> 10
0.56
0.57
0.39
5.50
0.15
2.87
> 10
1.86
0.58
0.18
1.99
4.28
1c
1d
1e
1f
H
CH=CHPh-4-Cl
(CH2)5Ph
H
1.53
1.00
1.46
0.051
ND
H
(CH2)7Ph
1g
1h
1i
OMe
OMe
H
(CH2)5Ph
(CH2)7Ph
Ph-4-t-Bu
CH2Ph
2a
2b
2c
2d
2e
2f
H
ND
H
CH2Ph-4-Ph
(CH2)4Ph
0.31
1.99
2.25
0.37
0.52
ND
H
H
(CH2)5Ph
H
(CH2)7Ph
H
Ph-4-t-Bu
CH2Ph-4-Ph
CH=CHPh-4-Cl
(CH2)5Ph
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
H
H
ND
H
ND
H
(CH2)7Ph
ND
H
Ph-4-t-Bu
Ph-4-Ph
ND
H
3.97
ND
H
CH2Ph
H
(CH2)2Ph-4-OH
CH2Ph-4-Ph
(CH2)4Ph
ND
H
ND
H
ND
H
(CH2)5Ph
ND
4g
5a
5b
H
Ph-4-t-Bu
Ph-4-t-Bu
ND
OMe
ND
< 10
< 10
ND
ND
1.95
> 10
> 10
6
7
0.041

<10
<10
ND
ND
>10
0.79 (0.021)d
2.92 (0.0091)d
13
14
9.10
54.6
64.7
0.66
0.17
1.21
0.26
> 10
> 10
15
16
< 10
< 10
ND
ND
0.19 (0.27)e
> 10
8.0f
SB-366791
AA-5-HT
0.27f
< 10
ND
1.0f
3.36f
OMDM-198
a Data are means of n = 4 separate determinations. Standard errors are not shown for the sake of clarity and were never higher than 10% of the means.
b As percent of ionomycin (4 M). ND, not determined when efficacy is lower than 10%.
c Determined against the effect of capsaicin (0.1 M).
d Data from ref. 5.
e Data from ref. 19h.
f Data from ref. 20b.
presence of an aliphatic chain (compounds 1a,b) was detrimental
for this activity. Boronic acids 13 and 14 were reported by
Minkkilä et al.5 to inhibit FAAH in the low nanomolar range
(IC50 = 21 and 9.1 nM, respectively), while the corresponding
values recorded by us were one and three orders of magnitude
higher (0.79 and 2.92 M, respectively). FAAH assays were
however performed at different pH values (7.4 by Minkkilä et
al.,5 and FAAH optimal pH = 9 by us) and it is well known that
pharmacological properties of FAAH are highly pH-dependent.27
In particular, rat brain hydrolysis of [3H]AEA shows an optimum
around pH 8-9.
nociception, qualify them as promising antinociceptive,
antihyperalgesic, and antioedemic agents in chronic and
inflammatory pain preclinical studies.
Acknowledgement
This research was supported by Sapienza Università di Roma
(Grant No C26A14TLFT).
References and notes
As concerns TRPV1 assays, benzylic amides 1 and benzylic
reverse amides 2 exhibited an overall superiority over their aryl
counterparts 3 and 4, and ureas 5 and 6. A reversed trend was
however observed with the 4-chlorostyryl and 4-t-butylphenyl
moieties (compare compounds 1e and 3c; 2a, 3a, and 4a; 5a and
6). The EC50 and IC50 values were, with the exception of 2c, 3c,
4a,b, 5a,b, 6, and 7, comparable and thus, only 3c, 4a, 5a,b, 6,
and 7 behaved as 'true' antagonists, while 2c and 4b acted as
weakly desensitizing agonists. The boronic functional group was
fully compatible with a TRPV1 modulating action but not
essential for it, as demonstrated by the comparison between
compounds 2f and 15,16, and 7 and SB-366791, and the
inactivity of compounds 1e, 3d,e, 4c,e,f,g, 13, and 14.
Thirteen of the compounds examined, that is, 1c,f-i, 2c-f, 3c,
4a,b, 5a,b targeted both FAAH and TRPV1 channel. In
particular, four of them, that is, 3c, 4a, 5a,b acted as dual
FAAH/TRPV1 blockers, the first three compounds comparing
favorably in terms of potency with the other chemotypes so far
presented by us.20 The ability of these compounds to inhibit
FAAH and activate/desensitize or directly antagonize TRPV1,
i.e. to target simultaneously two distinct targets involved in
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chromatography (silica gel, hexane/AcOEt mixtures). Sodium
periodate (3 mmol) was added at room-temperature to a solution
of the purified pinacol boronate ester 9 (1.0 mmol) in THF/H2O
(15/5 mL) and then 2 N HCl (0.40 mL) was added. The solution
was stirred at room temperature for 3-5 h, concentrated under
vacuum, diluted with water, and extracted with AcOEt. The
organic phase was separated, washed twice with water, dried
(Na2SO4), and evaporated under vacuum. The residue was
crystallized from THF/H2O to afford pure title compound. General
procedure for the synthesis of arylboronic acids 2 and 4. To a
stirred solution of the carboxylic acid 10 (1.0 mmol) in DMF (1
mL) were added at 0 °C HOBt (1.0 mmol) and EDC (1.0 mmol).
The mixture was stirred for 15 min at 0 °C and for 1 h at room
temperature. Then the appropriate amine (1.0 mmol) was 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
(silica gel, hexane/AcOEt mixtures). Sodium periodate (3 mmol)
was added at room-temperature to a solution of the purified
pinacol boronate ester 11 (1.0 mmol) in THF/H2O (15/5 mL) and
then 2 N HCl (0.40 mL) was added. The solution was stirred at
room temperature for 3-5 h, concentrated under vacuum, diluted
with water, and extracted with AcOEt. The organic phase was
separated, washed twice with water, dried (Na2SO4), and
evaporated under vacuum. The residue was crystallized from
THF/H2O to afford pure title compound. General procedure for
the synthesis of arylboronic acids 5 and 6. A solution of 4-tert-
butylphenyl isocyanate (1.0 mmol), pinacol boronate esters amine
hydrochloride or trifluoroacetate 8 (1.0 mmol) and Et3N (1.2
mmol) in dry DMF (5 mL) was stirred 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 (silica gel, hexane/AcOEt
mixtures). Sodium periodate (3 mmol) was added at room-
temperature to a solution of the purified pinacol boronate ester
urea 12 (1.0 mmol) in THF/H2O (15/5 mL) and then 2 N HCl
(0.40 mL) was added. The solution was stirred at room
temperature for 3-5 h, concentrated under vacuum, diluted with
water, and extracted with AcOEt. The organic phase was washed
twice with water, dried (Na2SO4), and evaporated under vacuum.
The residue was crystallized from THF/H2O to afford pure title
compound 5. Data for selected compounds: compound 3c: mp >
230°C; IR 3302, 1661, 1626, 1589, 1524, 1340, 1242, 1124, 1013,
972, 817 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 6.86 (1H, d, J =
16.0 Hz), 7.52 (2H, d, J = 8.4 Hz), 7.59 (1H, d, J = 16.0 Hz), 7.66
(4H, m), 7.77 (2H, d, J = 8.4 Hz), 7.94 (2H, s), 10.25 (1H, s); 13C
NMR (100 MHz, DMSO-d6) δ 117.95, 123.03, 128.98, 129.33,
133.60, 134.13, 134.84, 138.77, 140.71, 163.29. Compound 4a:
mp > 230 °C; IR 3349, 2959, 1656, 1638, 1517, 1407, 1320, 1251,
1100, 1008, 835, 729 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 1.28
(9H, s), 7.36 (2H, d, J = 8.8 Hz); 7.70 (2H, d, J = 8.8 Hz), 7.93
(4H, s), 8.26 (2H, s), 10.19 (1H, s); 13C NMR (100 MHz, DMSO-
d6) δ 31.18, 34.00, 120.15, 125.15, 126.44, 133.95, 136.19,
136.52, 145.99, 165.43. Compound 5a: mp > 230 °C. IR 3319,
2959, 1639, 1598, 1550, 1408, 1359, 1231, 1114, 1016, 816 cm-1;
1H NMR (400 MHz, DMSO-d6) δ 1.24 (9H, s), 4.30 (2H, d, J =
6.0 Hz), 6.54 (1H, t, J = 6.0 Hz), 722-7.33 (6H, m), 7.52 (2H, d, J
= 7.8 Hz), 8.00 (2H, s), 8.46 (1H, s); 13C NMR (100 MHz,
DMSO-d6) δ 31.17, 33.69, 42.66, 117.45, 125.10, 125.95, 134.07,
137.70, 142.14, 143.24, 155.19.
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on the enzymatic hydrolysis of anandamide was obtained using
membranes prepared from rat brain, incubated with the test
compounds and [14C]AEA (2.4 µM) in 50 mM Tris-HCl, pH 9, for
30 min at 37 °C. [14C]Ethanolamine produced from [14C]AEA
hydrolysis was measured by scintillation counting of the aqueous
phase after extraction of the incubation mixture with 2 volumes of
CHCl3/CH3OH = 2/1 (by volume). Data are expressed as the
concentration exerting 50% inhibition of AEA hydrolysis (IC50),
calculated by GraphPad. TRPV1 channel assays. HEK293 (human
embryonic kidney) cells stably over-expressing recombinant
human TRPV1 were grown on 100 mm diameter Petri dishes as
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7. To a stirred solution of the appropriate carboxylic acid (1.0
mmol) in DMF (1 mL) were added at 0 °C HOBt (1.0 mmol) and
EDC (1.0 mmol). The mixture was stirred for 15 min at 0 °C and
for 1 h at room temperature. Then, the pinacol boronate ester
amine hydrochloride or trifluoroacetate 8 (1.0 mmol) and Et3N
(1.0 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

mM glutamine, and maintained at 5% CO2 at 37 °C. The effect of
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determined by using the selective intracellular fluorescent probe
Fluo-4. On the day of the experiment, cells were loaded for 1 h at
room temperature with the methyl ester Fluo-4-AM (4 µM in
dimethyl sulfoxide containing 0.02% Pluronic F-127, Invitrogen)
in MEM without fetal bovine serum, then were washed twice in
Ty o ’ ff 145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.2
mM MgCl2, 10mM D-glucose, 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 changes in [Ca2+]i were determined
before and after the addition of various concentrations of test
compo y m a g c ll fl o c c λEX = 488 m, λEM
=
516 nm) at 25 °C. Curve fitting (sigmoidal dose–response variable
slope) and parameter estimation were performed with GraphPad
Prism® (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). The efficacy of the agonists was determined by
normalizing their effect to the maximum Ca2+ influx effect on
[Ca2+]i observed with application of 4 µM ionomycin (Cayman).
When significant, the values of the effect on [Ca2+]i in wild-type
(i.e., not transfected with any construct) HEK293 cells were taken
as baseline and subtracted from the values obtained from
transfected cells. Antagonist/desensitizing behaviour was
evaluated against capsaicin (0.1 µM)
, 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 Prism® 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
Bo f o ’ .
25. Prepared by reaction of HOBt/EDC-activated 2-phenylacetic acid
or 2-(4-hydroxyphenyl)acetic acid with 7-phenylheptan-1-amine.
26. Gunthorpe, M. J.; Rami, H. K.; Jerman, J. C.; Smart, D.; Gill, C.
H.; Soffin, E. M.; Hannan, S. L.; Lappin, S. C.; Egerton, J.; Smith,
G. D.; Worby, A.; Howett, L.; Owen, D.; Nasir, S.; Davies, C. H.;
Thompson, M.; Wyman, P. A.; Randall, A. D.; Davis, J. B.
Neuropharmacology 2004, 46, 133.
27. Holt, S.; Nilsson, J.; Omeir, R.; Tiger, G.; Fowler, C. J. Br. J.
Pharmacol. 2001, 133, 513.

Graphical Abstract
Arylboronic acids as dual-action FAAH and TRPV1
ligands
Leave this area blank for abstract info.
Enrico Morera,* Vincenzo Di Marzo, Ludovica Monti, Marco Allarà, Aniello Schiano Moriello, Marianna
Nalli, Giorgio Ortar, Luciano De Petrocellis*