J Med Chem Appendino et al

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Conformationally Constrained Fatty Acid Ethanolamides as Cannabinoid and Vanilloid
Receptor Probes
Giovanni Appendino,*
,†
Alessia Ligresti,
‡
Alberto Minassi,
†
Maria Grazia Cascio,
§
Marco Allara`,
‡
Orazio Taglialatela-Scafati,
|
Roger G. Pertwee,
§
Luciano De Petrocellis,
⊥
and Vincenzo Di Marzo*
,‡
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, UniVersita` del Piemonte Orientale, NoVara, Italy,
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, CNR Pozzuoli (NA), Italy, Institute of Medical Sciences, UniVersity of
Aberdeen, Aberdeen, Scotland, Endocannabinoid Research Group, Istituto di Cibernetica, CNR Pozzuoli, Italy, and Dipartimento di Chimica
delle Sostanze Naturali, UniVersita` di Napoli “Federico II”, Napoli, Italy
ReceiVed February 3, 2009
To investigate if certain acylethanolamides bind to both cannabinoid (CB1and CB2) and vanilloid TRPV1
receptors because of their conformational flexibility, we introduced a methylene lock on their ethanolamine
“head”, thereby generating a cyclopropane ring with two stereogenic centers and chiral cis/trans diastereomers
with different topology of presentation to binding sites. After resolution by chiral-phase HPLC, diastereo-
and enantiopure arachidonoyl-, oleoyl-, and palmitoylcyclopropanolamides were tested in assays of CB1,
CB2, and TRPV1 activity. Diastereodifferentiation between pairs of cis-trans isomers was observed only
for TRPV1 activity, with poor enantiodifferentiation. Methylenation introduced (i) CB1receptor affinity in
oleoylethanolamide while increasing in a diastereoselective way its activity at TRPV1 and (ii) strong
diastereoselective activity at TRPV1, but not cannabinoid, receptors in the otherwise inactive palmitoyle-
thanolamide. These results show that the N-alkyl group of acylethanolamides has a different role in their
interaction with cannabinoid and vanilloid receptors and that acylcyclopropanolamides qualify as CB1/
TRPV1 “hybrids” of potential therapeutic utility.
Introduction
The discovery of specific receptors for ∆9-tetrahydrocannab-
inol (1, THC
a
), the psychotropic agent of hemp (Cannabis satiVa
L.), and for capsaicin (2a), the pungent principle of chilli pepper
(Capsicum annuum L.), sparked intense research activity aimed
at evaluating the possibility of developing therapeutic drugs
targeting these receptors for a host of pathological conditions
that include pain, inflammation, emesis, obesity, neurodegen-
eration, and cancer.
1-3
Endogenous agonists for cannabinoid
and capsaicin receptors [known as “endocannabinoids” and
“endovanilloids”, respectively
4,5
(Figure 1)] were also discov-
ered from the pool of fatty acid conjugates of biogenic
amines.
6-12
While the plant-derived ligands show little pro-
miscuity in their targets, endocannabinoids like arachidonoyle-
thanolamide (3a, anandamide) and arachidonoyldopamide (4,
NADA) also activate TRPV1 receptors and hence behave as
endovanilloids. Conversely, certain fatty acid vanillamides
(capsaicinoids) like arachidonoylvanillamide (2b, arvanil)
13
and
phenylcetyl)ricinoleoylvanillamide (2c, PhAR)
14
also target CB1
and CB2cannabinoid receptors. This promiscuity, together with
the occurrence of a functional cross-talk between TRPV1 and
CB1,
15-17
and their colocalization in several nervous tissues
18,19
suggest that the two systems are closely intertwined in terms
of function and regulation and that synthetic dual CB1/TRPV1
agonists have the potential to correct dysregulation of both
systems better than agents individually targeting either TRPV1
or cannabinoid receptors.
2,3
Endocannabinoids and endovanilloids are modular com-
pounds in which a polar ethanolamine or dopamine head is
conjugated to a fatty acid. At the level of endogenous ligands,
TRPV1 channels can accept both mono- and polyunsaturated
acyl moieties
12,20
while cannabinoid receptors are more selective
and prefer polyunsaturated acyl moieties. Since cannabinoid and
vanilloid receptors are structurally unrelated, as they belong to
the families of G-protein-coupled receptors and of transient
receptor potential channels, respectively, it seems reasonable
to assume that the binding sites of endocannabinoids and
endovanilloids are different, and that the promiscuous behavior
of these ligands is due to their conformational flexibility, which
makes it possible for them to fit into distinct binding regions of
their target proteins. To test this hypothesis, we have focused
on the ethanolamide polar head of anandamide (3a), the
archetypal hybrid TRPV1-CB1ligand, and locked the relative
orientation of its hydroxyl and amido functions by inserting a
methylene bridge between the two methylene carbons. A similar
maneuver was carried out also on oleoylethanolamide (3b,
OEA), another endogenous TRPV1 ligand with little or no
activity at cannabinoid receptors, and palmityoletanolamide (3c,
PEA), an ethanolamide lacking direct activity at either receptors.
By insertion of a methylene lock, the polar head becomes chiral,
making it possible also to address the issue of chiral discrimina-
tion in the binding of these compounds. In fact, while endocan-
nabinoids and endovanilloids are achiral (Figure 1), THC and
* To whom correspondence should be addressed. For G.A.: phone,
+390321375744; fax: +390321375621; e-mail, Giovanni.Appendino@
pharm.unipmn.it. For V.D.: phone, 39 81 867 5093; fax, 39 081 80 41
770; e-mail, vdimarzo@icmib.na.cnr.it.
†
Universita` del Piemonte Orientale.
‡
Istituto di Chimica Biomolecolare.
§
University of Aberdeen.
|
Universita` di Napoli “Federico II”.
⊥
Istituto di Cibernetica.
a
Abbreviations: AEA, arachidonoylethanolamide; CC, column chro-
matography; AcOH, acetic acid; DCM, dichloromethane; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DMAP, 4-dimethylaminopyridine; EDCI,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; EtOAc,
ethyl acetate; EtOH, ethanol; Et2Zn, diethylzinc; HPLC, high performance
liquid chromatography; NADA, N-arachidonoyldopamine; OEA, oleoyle-
thanolamide; PEA, palmitoylethanolamide; TBAF, tetrabutylammonium
fluoride; TBDMS, tert-butyldimethylsilyl; THC, ∆9-tetrahydrocannabinol;
TRPV1, transient receptor potential vanilloid type-1.
J. Med. Chem. 2009, 52, 3001–3009 3001
10.1021/jm900130m CCC: $40.75 2009 American Chemical Society
Published on Web 04/10/2009

several potent synthetic CB1and CB2ligands interact with
cannabinoid receptors in an enantioselective way.
1,21
Results
Synthesis of the Racemic Cyclopropanolamides (()-9a-c
and (()-10a-c. Racemic cyclopropanolamides were prepared
by the stereoselective Simmons-Smith cyclopropanation
22
of
the E/Zdiastereomeric silyl enol ethers 6a and 6b, obtained as
an easily separable mixture by enolization and silyl trapping of
the known N-phtaloylaminoaldehyde 5(see Experimental Sec-
tion). Removal of the phtaloyl moiety by hydrazinolysis afforded
a silyl protected cyclopropanolamine that was not isolated but
directly acylated with the DCC-DMAP protocol and next
desilylated (Figure 2).
Resolution of the Racemic Arachidonoyl- and Oleoylcyclo-
propanolamides (()-9a,b and (()-10a,b and Assignment of
the Absolute Configuration of the Enatiomerically Pure Cy-
clopropanolamides. The four racemates were resolved by
preparative chiral HPLC chromatography. Two distinct chro-
matographic stationary phases (i.e., a Chirex-3020, Phenomenex,
column containing a stationary phase made with either S-tert-
leucine or S-valine and a R-1-R-naphthylethylamine urea
linkage) had to be employed to separate the cis and the trans
racemates. In all cases, an isocratic elution mode was employed.
The absolute configuration of the enantiomerically pure cyclo-
propanolamides (shown in Figure 3) was established by ap-
plication of a modified Mosher method
23
(Figure 4).
Affinity of the Cyclopropanolamides for Cannabinoid Re-
ceptors. The two diastereomeric arachidonoylcyclopropanola-
mides rac-9a and rac-10a did not exhibit affinity for both
cannabinoid receptors higher than that of anandamide, their
affinity at CB1receptors being always higher than that at CB2
receptors. No difference between the trans and cis diastereomers
was observed for CB1receptors, while the cis diastereomer rac-
10a was slightly more active than its trans diastereomer rac-
9a at the CB2receptor (Table 1). After chiral phase HPLC
separation, however, while the 13a and 14a enantiomers
exhibited similar affinity for CB1or CB2receptors, the 12a
enantiomer showed slightly higher affinity than its 11a enan-
tiomer for both receptors (Table 2). In sharp difference with
OEA, the oleoylcyclopropanolamides rac-9b and rac-10b could
bind with reasonably good affinity both cannabinoid receptors
(Table 1). The diastereoisomeric oleoylcyclopropanolamides
rac-9b and rac-10b exhibited all similar affinities for CB1or
CB2receptors, although within the enantiopure compounds
11b-14b, a trend for a higher affinity toward CB1receptors
was observed (Table 2). Finally, the diasteroemeric palmitoyl-
cyclopropanolamides rac-9c and rac-10c exhibited no affinity
for cannabinoid receptors and therefore were not further resolved
(Table 1).
Functional Activity of N-Arachidonoylcyclopropanolamides
at CB1Receptors. Two of the compounds with the highest
affinity at CB1receptors, i.e., compounds 11a and 12a, were
also tested for their ability to stimulate [35S]GTPγS binding to
mouse brain membranes. In this assay, agonists, by activating
the coupling of G-protein-coupled receptors to the R-subunits
of the G-protein, will stimulate the binding of [35S]GTPγSto
cell membranes containing the respective receptors. Inverse
agonists will do the opposite, and neutral antagonist will not
exert any effect on [35S]GTPγS binding. In our case, both 11a
and 12a behaved as partial agonists (EC50 )2.55 and 0.78 µM
and Emax )47.2% and 39.8%, respectively, compared to the
CB1/CB2full agonist WIN55,212-2, Emax )100%), albeit with
potency and efficacy lower than that of anandamide tested under
the same conditions (EC50 )0.36 µM and Emax )68.6%)
(Figure 5).
Activity of Cyclopropanolamides at TRPV1 Receptors.
Within the two racemic cis/trans arachidonoylcyclopropanola-
mides, only the trans-diastereomer rac-9a retained an activity
similar to or even higher than that of anandamide at elevating
intracellular Ca2+in HEK-293 cells stably transfected with the
human recombinant TRPV1 channel. Conversely, the cis-
diastereoisomer rac-10a was significantly less potent than
anandamide (Table 1). A further significant, although less
dramatic, dissection of the pharmacological activity was also
observed with the two enatiopure cis- enantiomers 13a and 14a,
but little enantiodifferentiation was observed within the more
active trans-enantiomers 11a and 12a. Thus, the 13a enantiomer
was 3-fold more potent than the 14a enantiomer (Table 2 and
Figure 6). Similar, although not identical, results were obtained
with the cyclopropanolamides of oleic acid. In this case, the
trans-diastereoisomer rac-9b was more than 50-fold more potent
than OEA, while the cis-diastereoisomer rac-10b was equipotent
with OEA (Table 1). Furthermore, the two trans-enantiomers
11b and 12b, just like their corresponding cis-enantiomers 13b
and 14b, exhibited similar potency, although the former was
10-fold more potent than the latter (Table 2 and Figure 6).
However, although the enantiomers 13b and 14b exhibited
similar potency, the former was still more efficacious than the
latter. Finally, despite the almost total lack of direct activity of
palmitoylethanolamide at TRPV1 receptors, the trans-palmi-
Figure 1. Chemical structures of plant-derived, endogenous, and
synthetic agonists of cannabinoid and TRPV1 receptors.
3002 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 Appendino et al.

toylcyclopropanolamide rac-9c was as potent as capsaicin
(although less efficacious) at elevating intracellular Ca2+via
these channels in HEK-293 cells, whereas its cis-diastereoisomer
rac-10c was almost inactive (Table 1).
Discussion and Conclusions
Conformational constraining is a popular strategy to identify
the active conformation of flexible ligands, and this approach
has been successfully applied in various fields of medicinal
chemistry, including peptidomimetic structures.
24
In ethanola-
mides, rotation around the sp3carbon-carbon bond of the polar
head interconverts hydrogen-bonded syn and non-hydrogen-
bonded anti rotamers (Figure 7). The H-bonded syn rotamers
are expected to prevail in a relatively apolar binding pocket,
whereas their fitting into a cavity rich with H-bonding elements
would favor the anti-conformation, which can establish more
H-bonding contacts with a biological surface. The observation
that the presence of the hydroxyl is redundant for the cannab-
inoid receptor activity of anandamide but is critical for its
activation of TRPV1 channels
25
suggests that different binding
modes are involved for the two receptors. To address this issue,
we have introduced a methylene bridge around the central bond
of the ethanolamine moiety, thereby transforming fleeting syn/
anti rotamers into noninterconverting diastereomeric cis/trans
isomers (Figure 7, parts A and B, respectively). Our results on
the conformationally constrained analogues of anandamide fully
confirm this. Thus, the much higher TRPV1-stimulating activity
of the trans-isomers of methylenandandamide compared to their
cis isomers suggests that TRPV1 prefers to bind to the anti-
conformation of anandamide. By contrast, the very similar
cannabinoid receptor affinity of the cis and trans isomers
suggests that the terminal oxymethylene is not involved in the
binding to CB1and CB2, an observation that is in full accordance
with the lack of requirement of the primary hydroxyl of
anandamide for its binding to these receptors.
26
In fact, the
N-alkyl group of anandamide might simply act as a conforma-
tional bias to maintain the amide carbonyl and the acidic N-H
amide bond in the reciprocal geometry necessary for optimal
binding to cannabinoid receptors.
26
Methylenation of OEA yielded surprising results, since it
strongly increased the affinity for CB1and CB2receptors, which
is otherwise marginal in the parent compound, while also
potentiating, in a diastereoselective way, the efficacy and
potency at TRPV1. A similar potentiation of the vanilloid
activity was also observed in PEA. In all cases, the trans isomers
were more active at TRPV1 than the cis isomers, confirming
the findings with anandamide.
In all cases, little enantiodifferentiation was observed within
ligands, both for the binding to cannabinoid receptors and for
TRPV1 functional activity. However, some statistically signifi-
cant enantioselectivity in terms of either potency or efficacy at
TRPV1 channel activity was observed within the less active
cis diastereoisomers of both arachidonoyl- and oleoylcyclopro-
panolamides, in both cases with enantiomer 13 (Figure 3)
exhibiting the highest activity.
The present observations have several potentially important
and previously unsuspected implications. First of all, we have
shown for the first time that also TRPV1 receptors, like other
receptors, can “sense” configurational differences in their
agonists. This observation is likely to open new studies on the
molecular organization of the TRPV1 binding site and, in
particular, on the role of specific amino acid residues in the
recognition of anandamide. This compound binds to TRPV1 at
the same site as capsaicin,
27
and our observations should assist
in the identification of the residues that recognize the ethanol-
amine “head” of anandamide and OEA.
28
Second, we have
demonstrated that a reduction of the flexibility of the ethanol-
amine “head” of an unsaturated and a monounsaturated acyle-
thanolamide can dramatically improve their otherwise weak
activity at TRPV1 receptors, provided that the cyclopropyl group
has a trans-configuration. This chemical modification has such
Figure 2. Synthesis of the racemic cyclopropanolamides (()-9a-cand (()-10a-c.
Figure 3. Absolute configuration of the enantiopure N-acylcyclopro-
panolamides resolved by chiral preparative HPLC.
Fatty Acid Ethanolamides as Probes Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3003

a dramatic effect that the trans-diastereoisomers of these
compounds and of anandamide, which already exhibits activity
at TRPV1 receptors, become as potent and, in most cases, as
efficacious as capsaicin. These compounds represent the first
potent TRPV1 agonists that lack a phenolic (vanillyl or
catecholic) group in their polar head. The effect of cyclopro-
panation on TRPV1 activity was also evident in the activity of
the methylene derivatives of PEA, which represent the first
example of TRPV1 agonists with unfunctionalized linear acyl
chain. With OEA, but not with anandamide, the introduction
of the cyclopropyl group also enhanced the affinity for CB2and,
especially, CB1receptors, although in a nondiastereoselective
way. This was somewhat surprising, since, although the en-
docannabinoids are achiral, enantiodifferentiation in binding to
cannabinoid receptors was shown in optically active ananda-
mide
29
and 2-arachidonoylglycerol
30
analogues.
Taken together, our observations qualify conformational
constraint as a critical maneuver
31
to map the binding site of
ethanolamides to cannabinoid receptors. Finally, our findings
might also have important therapeutic implications, since we
have identified the enantiomeric trans-methylenoleamides 11a
and 12a as potent CB1/TRPV1 “hybrid” agonists worth further
investigation as potent analgesic, antiemetic, neuroprotective,
and antitumor leads.
13,32-35
Compared to arvanil, the best
documented CB1/TRPV1 “hybrid”, 11a and 12a, are endowed
with a better balance between the two types of activity that are
instead biased toward TRPV1 for arvanil.
36
Clearly, dual CB1/
TRPV1 agonists are likely to correct dysregulation of endocan-
nabinoid and endovanilloid signaling systems better than agents
individually targeting either TRPV1 or CB1receptors.
2,3
Possibly
for this reason, compounds like arvanil were found to elicit
beneficial effects in animal models of pain, neurotoxicity, and
emesis that were stronger than those of “single” CB1and TRPV1
agonists tested under the same conditions.
32-34
On the other
hand, the development of CB1/TRPV1 “hybrids” should pose
less toxicological and/or pharmacokinetic problems than the
coadministration of “single” CB1and TRPV1 agonists.
In conclusion, our results show that the ethanolamine moiety
has a different role in the activation of vanilloid and cannabinoid
receptors by their endogenous ligand anandamide. For the first
time, a diastereomeric dependence for TRPV1 activation by its
ligands has been observed, identifying the anti-rotamer of
acylethanolamides as their active conformation and obtaining
compounds the potency of which rivals that of capsaicinoids.
Finally, we have presented evidence that methylenation of
ethanolamides can dramatically potentiate the activity of non-
arachidonoyl homologues of this family of lipids at TRPV1 and,
at least for oleoyl derivatives, also at cannabinoid receptors.
Taken together, these findings have potentially important
implications for our understanding of the molecular mechanisms
involved in the activation of TRPV1 and cannabinoid receptors
and for their translation into the development of new therapeutic
drugs.
Experimental Section
1. General Synthetic Procedures. For gravity column chroma-
tography (CC), Merck silica gel (70-230 mesh) and Macherey-
Nagel aluminum oxide neutral were used. Chiral-phase HPLC was
performed on a Knauer apparatus equipped with a refractive index
detector. For IR experiments, a Shimadzu DR 8001 spectropho-
tometer was used. For NMR experiments, Jeol Eclipse (300 and
75 MHz for 1H and 13C, respectively) and Varian INOVA (500
Figure 4. ∆δ(S-R) observed for the MTPA esters of 11a,13a,11b, and 13b.
Table 1. Effects of the Racemic Mixtures of the Two Diastereoisomers
of Each N-Acylcyclopropanolamide Synthesized Here on Cannabinoid
and TRPV1 Receptors
a
a
Affinity of compounds for human recombinant CB1and CB2receptors
was evaluated using membranes from HEK-293 cells transfected with the
respective cDNAs and [3H]CP55,940 as the high affinity ligand and WIN
55212-2 as the heterologous competitor for nonspecific binding. Kivalues
are calculated by applying the Cheng-Prusoff equation to the IC50 values
for the displacement of the bound radioligand by increasing concentrations
of the test compound. Data are reported as mean values (SEM of at least
n)3 experiments. The functional activity of compounds at human
recombinant TRPV1 receptors was evaluated by measuring the effect on
intracellular Ca2+in HEK-293 cells stably transfected with the cDNA
encoding for the receptor. Data are the mean values (SEM of at least n
)3 experiments. The effects of standard compounds is also shown for
comparison. The data on OEA and PEA affinity for cannabinoid receptors
and functional activity at TRPV1 receptors are in agreement with previously
published data.
28,40,41
3004 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 Appendino et al.

and 125 MHz for 1H and 13C, respectively) spectrometers were used
and chemical shifts are reported in δvalues. Benzene, CH2Cl2, and
THF by filtration over alumina prior were used directly in the
reaction flask. Petroleum ether refers to the fraction boiling between
40 and 60 °C. All reactions were conducted under nitrogen in dry
solvents unless stated otherwise. Monitoring by TLC was done on
Merck 60 F254 (0.25 mm) plates, which were visualized by UV
inspection and/or staining with 5% H2SO4in ethanol and heating.
Organic phases were dried with Na2SO4before evaporation. ESI
MS data were taken on a LCQ Finnigan MAT apparatus, in the
positive ion mode. A Knauer HPLC apparatus equipped with
refraction index detector was used to assess purity (>95%) of all
final products. Chirex-3014 or Chirex-3020 (Phenomenex) columns
were used, with elution with EtOAc/n-hexane mixtures and 0.7 mL/
min as flow rate.
(E)-(Z)-2-[2-(tert-Butyldimethylsilyloxy)ethenyl]isoindoline-1,3-
dione (6a and 6b). To a stirred solution of (1,3-dioxo-1,3-dihydro-
2H-isoindol-2-yl)acetaldeide (5) (420 mg, 2.22 mmol) in dry CH2Cl2
(7 mL), TBDMS triflate (997 µL, 4.36 mmol, 1.9 mol. equiv) and
DBU (1.081 mL, 7.23 mmol, 3.25 mol equiv) were added.
37
The
mixture was stirred for 15 min at room temperature and then worked
up by the addition of2NH
2SO4and dilution with CH2Cl2. The
organic phase was dried and evaporated, and the residue was
purified by gravity CC on silica gel (12.5 g, petroleum ether-EtOAc,
95:5, as eluant) to afford 250 mg (37%) of the Eisomer (6a) and
265 mg (39%) of the Zisomer (6b). Because of their instability,
both compounds were used without further separation for the
methylenation step. E-Isomer (6a): yellowish oil. 1H NMR (300
MHz, CDCl3): δ7.78 (2H, m), 7.67 (2H, m), 7.50 (1H, d, J)
11.3 Hz), 6.40 (1H, d, J)11.3 Hz), 0.95 (9H, s), 0.19 (6H, s).
Z-Isomer (6b): yellowish oil. 1H NMR (300 MHz, CD3COCD3): δ
7.86 (4H, m), 6.60 (1H, d, J)4.3 Hz), 5.41 (1H, d, J)4.3 Hz),
0.85 (9H, s), 0.17 (6H, s). 13C NMR (CDCl3, 75 MHz): δ166.8
(s), 141.0 (d), 133.9 (d), 132.4 (s), 123.3 (d), 99.2 (d), 25.4 (q),
-5.3 (q).
(trans/cis)-2-((()-2-(tert-Butyldimethylsilyloxy)cyclopropyl)-
isoindoline-1,3-dione (7a and 7b). To a cooled (ice bath) stirred
solution of 6a (610 mg, 2 mmol) in dry benzene, Et2Zn (1 M
solution in hexane, 30 mL, 30 mmol, 15 mol equiv) and CH2I2
(2.2 mL, 30 mmol, 15 mol equiv) were added. The mixture was
stirred overnight at 65 °C and then worked up by the addition of 2
NH
2SO4and dilution with petroleum ether. The organic phase was
washed with saturated NaHCO3, dried, evaporated, and the residue
was purified by gravity CC on silica gel (18 g, petroleum
ether-EtOAc, 95:5, as eluant) to afford 310 mg (49%) of 7a as
yellow oil. 1H NMR (300 MHz, CDCl3): δ7.79 (2H, m), 7.69 (2H,
m), 3.90 (1H, m), 2.79 (1H, m), 1.27-1.19 (2H, m), 0.91 (9H, s),
0.21 (6H, s). 13C NMR (CDCl3, 75 MHz): δ168.5 (s), 134.0 (d),
Table 2. Effects of the Two Enantiomers of Each Racemic Mixture of N-Arachidonoyl- and N-Oleoylcyclopropanolamides on Cannabinoid and TRPV1
Receptors
a
a
Affinity of compounds for human recombinant CB1and CB2receptors was evaluated using membranes from HEK-293 cells transfected with the respective
cDNAs and [3H]CP55,940 as the high affinity ligand and WIN 55212-2 as the heterologous competitor for nonspecific binding. Kivalues are calculated by
applying the Cheng-Prusoff equation to the IC50 values for the displacement of the bound radioligand by increasing concentrations of the test compound.
Data are reported as mean values (SEM of at least n)3 experiments. The functional activity of compounds at human recombinant TRPV1 receptors was
evaluated by measuring the effect on intracellular Ca2+in HEK-293 cells stably transfected with the cDNA encoding for the receptor. Data are mean values
(SEM of at least n)3 experiments. Efficacy was evaluated in comparison to the effect on intracellular Ca2+by ionomycin (4 µM).
Figure 5. Effect of (a) 11a (n)8), (b) 12a (n)8), and (a, b) anandamide (n)12) on the level of [35S]GTPγS binding to mouse brain
membranes. Each symbol represents the mean percent increase in [35S]GTPγS binding (SEM. WIN55,212-2 was taken as the full agonist (Emax
)100%). As a reference compound, CP55,940 behaved as a more efficacious (Emax )83%) and potent (EC50 )10 nM) agonist in this assay.
Fatty Acid Ethanolamides as Probes Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3005

131.8 (s), 123.2 (d), 51.3 (d), 28.8 (d), 25.8 (q), 15.5 (t), -4.7 (q),
-5.1 (q). CI-EIMS: m/z[M +H]+360 [C20H29NO3Si +H]+. Under
similar conditions, 6b (335 mg, 1.1 mmol) afforded 326 mg (93%)
of 7b as yellow oil. 1H NMR (300 MHz, CDCl3): δ7.78 (2H, m),
7.67 (2H, m), 3.63 (1H, m), 2.61 (1H, m), 1.48 (1H, m), 1.22 (1H,
m), 0.66 (9H, s), 0.01 (3H, s) 0.00 (3H, s). 13C NMR (CDCl3,75
MHz): δ169.3 (s), 133.8 (d), 132.0 (s), 122.9 (d), 48.2 (d), 25.4
(q), 25.2 (d), 12.4 (t), -5.1 (q). CI-EIMS: m/z[M +H]+360
[C20H29NO3Si +H]+.
(()-trans-N-(2-Hydroxycyclopropyl)acylamides 9a-c. Synthesis
of 9a as Representative. To a stirred solution of 7a (422 mg, 1.37
mmol) in CH2Cl2(5 mL) and EtOH (1 mL), NH2NH2·H2O (440
µL, 8.26 mmol, 6 mol equiv) was added. The mixture was stirred
for4hatroom temperature, and then filtered over a Celite pad.
The organic phase was evaporated to afford 8a, directly used for
the acylation step. To this purpose, to a stirred solution of crude
8a (175 mg, 0.93 mmol) in dry CH2Cl2(2.5 mL), arachidonic acid
(305 µL, 0.93 mmol, 1 mol equiv), EDCl (356 mg, 1.86 mmol, 2
mol equiv), and catalyst DMAP were added. The mixture was
stirred under nitrogen at room temperature overnight and then
worked up by evaporation. The residue was adsorbed on silica gel
and filtered by gravity CC on neutral alumina (5 g, petroleum
ether-EtOAc, 95:5, as eluant) to afford 330 mg of the protected
amide. The latter was dissolved in dry THF (5 mL), and AcOH
(40 µL, 1.07 mmol, 1.5 mol equiv) and TBAF (1 M in THF, 1.07
mL, 1.07 mmol, 1.5 mol equiv) were added. The solution was stirred
under nitrogen at room temperature for 10 min and then worked
up by the addition of brine and dilution with EtOAc. The organic
phase was dried, evaporated, and the residue was purified by gravity
CC on silica gel (8 g, petroleum ether-EtOAc, 5:5, as eluant) to
afford 145 mg (29% from 7a)of9a as a pale-yellow oil.
Compounds rac-9b and rac-9c were obtained in the same way from
7a, using oleic and palmitic acid, respectively, in the acylation step
(31% and 24% overall yield, respectively).
(()-trans-N-(2-Hydroxycyclopropyl)palmitamide (9c). Colorless
foam. 1H NMR (300 MHz, CDCl3): δ5.38 (1H, br s, NH), 3.39
(1H, m, H-2), 2.67 (1H, m, H-1), 2.10 (2H, t, J)7.3 Hz, H-2′a,b,),
1.58 (2H, m, H-3′a,b), 1.24 (24H, br s, H2-4′a,b to H2-15′a,b), 1.09
(1H, m, H-3a), 0.87 (3H, t, J)6.4 Hz, H-16′), 0.76 (1H, m, H-3b).
13C NMR (CDCl3, 75 MHz): δ174.8 (s), 52.7 (d), 36.5 (t), 31.9
(t), 30.5 (d), 29.7 (t), 29.5 (t), 29.43 (t), 29.40 (t), 29.3 (t), 25.6 (t),
22.7 (t), 15.8 (t), 14.1 (q). CI-EIMS: m/z[M +H]+312 [C19H37NO2
+H]+.
(()-cis-N-(2-Hydroxycyclopropyl)acylamides 10a-c. Synthesis
of 10a as Representative. To a stirred solution of 7b (392 mg, 1.28
mmol) in CH2Cl2(5 mL) and EtOH (1 mL), NH2NH2·H2O (410
µL, 7.68 mmol, 6 mol equiv) was added. The mixture was stirred
for3hatroom temperature and then filtered over a pad of Celite.
The organic phase was evaporated to afford 8b that was directly
used for the acylation step. To this purpose, to a stirred solution of
crude 8b (160 mg, 0.85 mmol) in dry CH2Cl2(2.5 mL), arachidonic
acid (279 µL, 0.85 mmol, 1 mol equiv), EDCl (325 mg, 1.7 mmol,
2 mol equiv), and catalyst DMAP were added. The mixture was
stirred under nitrogen at room temperature overnight and then
worked up by evaporation. The residue was adsorbed on silica gel
and next vacuum-filtered on neutral alumina (5 g, petroleum
ether-EtOAc, 95:5, as eluant) to afford 360 mg of the crude
protected amide. Under a nitrogen atmosphere, the latter was
dissolved in dry THF (5 mL), and AcOH (45 µL, 1.2 mmol, 1.5
mol equiv) and TBAF (1 M in THF, 1.20 mL, 1.12 mmol, 1.5 mol
equiv) were then added. The solution was stirred under nitrogen at
room temperature for 10 min and next worked up by the addition
of brine and dilution with EtOAc. The organic phase was dried,
evaporated, and the residue was purified by gravity CC on silica
gel (8 g, petroleum ether-EtOAc, 5:5, as eluant) to afford 153 mg
(33% from 7b)of10a as a pale-yellow oil. Compounds rac-10b
and rac-10c were obtained in the same way from 7b, using oleic
and palmitic acid, respectively, in the acylation step (34% and 26%
overall yield, respectively).
(()-(cis)-N-(2-Hydroxycyclopropyl)palmitamide (7b). Amor-
phous foam. 1H NMR (300 MHz, CDCl3): δ5.76 (1H, bs, NH),
3.61 (1H, m, H-2), 2.60 (1H, m, H-1), 2.18 (2H, t, J)7.0 Hz,
H-2′), 1.61 (2H, m, H-3′a,b), 1.25 (24H, H-4′a,b to H2-15′a,b, m),
0.97 (1H, m, H-3a), 0.87 (3H, t, J)6.4 Hz, H-16′), 0.59 (1H, m,
H-3b). 13C NMR (CDCl3, 75 MHz): δ175.6 (s), 49.0 (d), 36.6 (t),
30.0 (t), 29.7 (t), 29.5 (t), 29.4 (t), 29.3 (t), 27.5 (d), 25.8 (t), 22.7
(t), 14.5 (t), 14.2 (q). CI-EIMS: m/z[M +H]+312 [C19H37NO2+
H]+.
Resolution of (()-9a and (()-10a by Chiral Phase HPLC. (a)
The racemic trans N-arachidonoylcyclopropanolamide (()-9a was
separated by chiral-phase HPLC with a Chirex-3020 (Phenomenex)
column (stationary phase with S-tert-leucine and R-1-R-naphthyl-
ethylamine urea linkage). The eluent mixture EtOAc/n-hexane, 65:
35, was used in isocratic mode with the flow 0.7 mL/min to obtain
the enantiomers 11a (1S,2S)(tR)7.8) and 12a (1R,2R)(tR)8.5)
(see below for the assignment of the absolute configuration).
(1S,2S)-N-Arachidonoylcyclopropanolamide (11a). Light-yellow
oil. [R]D
22 +5(c0.3, CHCl3). 1H NMR (500 MHz, CDCl3): δ5.71
(1H, d, J)5.0 Hz, NH), 5.35 (16H, m, H-5′a,b, H-6′a,b, H-8′a,b,
H-9′a,b, H-11′a,b, H-12′a,b, H-14′a,b, H-15′a,b), 3.35 (1H, m, H-2),
2.82 (6H, m, H-7′a,b, H-10′a,b, H-13′a,b), 2.65 (1H, m, H-1), 2.13
(2H, t, J)6.7 Hz, H-2′a,b), 2.06 (2H, q, J)6.0 Hz, H-4′a,b),
2.05 (2H, m, H-16′a,b), 1.70 (4H, m, H-3′a,b, H-17′a,b), 1.30 (4H,
m, H-18′a,b, H-19′a,b), 1.11 (1H, m, H-3a), 0.88 (3H, t, J)7.1
Hz, H-20′), 0.73 (1H, m, H-3b). 13C NMR (125 MHz, CDCl3): δ
175.3 (C-1′, s), 130.3 (C-5′, C-15′, d), 127.8 (C-6′, C-8′, C-9′, C-11′,
C-12′, C-14′, d), 52.4 (C-2, d), 36.3 (C-2′, t), 32.0 (C-18′, t), 31.6
(C-1, d), 30.0 (C-17′, t), 27.8 (C-16′, t), 27.7 (C-3′, t), 26.7 (C-4′,
t), 25.9 (C-3, t), 25.7 (C-7′, C-10′, C-13′, t), 23.1 (C-19′, t), 13.5
(C-20′, q). ESI-MS: m/z382 [M +Na]+.
Figure 6. Dose response curves for the four pure enantiomers of (a)
N-arachidonoylcyclopropanolamides 11a,12a,13a, and 14a and (b)
N-oleoylcyclopropanolamides 12b,11b,13b, and 14b. Experimental
conditions are described in the Experimental Section and in the
footnotes to Tables 1 and 2.
Figure 7. Syn (A) and anti (B) conformations of N-acylethanolamides.
3006 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 Appendino et al.

(1R,2R)-N-Arachidonoylcyclopropanolamide (12a). Light-yellow
oil. [R]D
22 -5(c0.3, CHCl3). For 1H NMR, 13C NMR, and MS
data, see those of 11a.
(b) The racemic cis-N-arachidonoylcyclopropanolamide (()-10a
was resolved by chiral-phase HPLC with a Chirex-3014 (Phenom-
enex) column (stationary phase with S-valine and R-1-R-naphth-
ylethylamine urea linkage). The eluent mixture EtOAc/n-hexane,
8:2, was used in isocratic mode with the flow 0.7 mL/min to obtain
the enantiomers 13a (1S,2R)(tR)4.1) and 14a (1R,2S)(tR)5.0)
(see below for the assignment of the absolute configuration).
(1S,2R)-N-Arachidonoylcyclopropanolamide (13a). Light-yellow
oil. [R]D
22 -5(c0.5, CHCl3). 1H NMR (500 MHz, CDCl3): δ5.80
(1H, d, J)5.0 Hz, NH), 5.37 (8H, m, H-5′, H-6′, H-8′, H-9′, H-11′,
H-12′, H-14′, H-15′), 3.62 (1H, m, H-2), 2.82 (6H, m, H-7′a,b,
H-10′a,b, H-13′a,b), 2.63 (1H, m, H-1), 2.20 (2H, t, J)6.7 Hz,
H-2′a,b), 2.10 (2H, q, J)6.0 Hz, H-4′a,b), 2.07 (2H, m, H-16′a,b),
1.72 (4H, m, H-3′a,b, H-17′a,b), 1.31 (4H, m, H-18′a,b, H-19′a,b),
1.00 (1H, m, H-3a), 0.88 (3H, t, J)7.1 Hz, H-20′), 0.58 (1H, m,
H-3b). 13C NMR (125 MHz, CDCl3): δ175.3 (C-1′, s), 130.3 (C-
5′, C-15′, d), 127.8 (C-6′, C-8′, C-9′, C-11′, C-12′, C-14′, d), 49.3
(C-2, d), 36.2 (C-2′, t), 32.0 (C-18′, t), 30.0 (C-17′, t), 28.2 (C-1,
d), 27.8 (C-16′, t), 27.7 (C-3′, t), 26.7 (C-4′, t), 26.3 (C-3, t), 25.9
(C-7′, C-10′, C-13′, t), 23.1 (C-19′, t), 13.5 (C-20′, q). ESI-MS:
m/z382 [M +Na]+.
(1R,2S)-N-Arachidonoylcyclopropanolamide (14a). Light-yellow
oil. [R]D
22 +5(c0.8, CHCl3). For 1H NMR, 13C NMR, and MS
data, see those of 13a.
2. Resolution of (()-9b and (()-10b by Chiral Phase HPLC.
(a) The racemic trans-N-oleoylcyclopropanolamide (()-9b was
separated by chiral-phase HPLC with a Chirex-3020 (Phenomenex)
column. The eluent mixture EtOAc/n-hexane, 55:45, was used in
isocratic mode with the flow 0.7 mL/min to obtain pure stereoi-
somers 11b (1S,2S)(tR)9.5) and 12b (1R,2R)(tR)8.5) (see
below for the assignment of the absolute configuration).
(1S,2S)-N-Oleoylcyclopropanolamide (11b). Gum. [R]D
22 +11 (c
0.2, CHCl3). 1H NMR (500 MHz Varian INOVA, CDCl3): δ5.87
(1H, d, J)5.0 Hz, NH), 5.32 (2H, m, H-9′, H-10′), 3.35 (1H, m,
H-2), 2.63 (1H, m, H-1), 2.10 (2H, t, J)6.7 Hz, H-2′a,b), 2.00
(4H, m, H-8′a,b, H-11′a,b), 1.58 (2H, m, H-3′a,b), 1.30 (20H, m,
H-4′a,b to H-7′a,b, and H-12′a,b to H-17′a,b), 1.09 (1H, m, H-3a),
0.87 (3H, t, J)7.1 Hz, H-18′), 0.75 (1H, m, H-3b). 13C NMR
(125 MHz Varian INOVA, CDCl3): δ175.2 (C-1′, s), 130.1 (C-9′,
C-10′, d), 52.9 (C-2, d), 36.1 (C-2′, t), 32.1 (C-4′to C-7′, C-12′to
C-16′, t), 30.6 (C-1, d), 29.6 (C-3′, t), 25.7 (C-3, t), 24.7 (C-8′,
C-11′, t), 23.0 (C-17′, t), 14.3 (C-18′, q). ESI-MS (LCQ Finnigan
MAT, positive ions) m/z360 [M +Na]+.
(1R,2R)-N-Oleoylcyclopropanolamide (12b). Amorphous foam,
[R]D
22 -11 (c0.2, CHCl3). For 1H NMR, 13C NMR, and MS data,
see those of 11b.
(b) The racemic cis-N-oleoylcyclopropanolamide 10b was
resolved by chiral-phase HPLC with a Chirex-3014 (Phenom-
enex) column (see above). The eluent mixture EtOAc/n-hexane,
65:35, was used in isocratic mode with the flow 0.7 mL/min to
obtain pure stereoisomers 13b (1S,2R)(tR)3.5) and 14b (1R,2S)
(tR)4.5) (see below for the assignment of the absolute
configuration).
(1S,2R)-N-Oleoylcyclopropanolamide (13b). Gum. [R]D
22 -4(c
0.2, CHCl3). 1H NMR (500 MHz Varian INOVA, CDCl3): δ5.87
(1H, d, J)5.0 Hz, NH), 5.33 (2H, H-9′, H-10′), 3.59 (1H, m,
H-2), 2.63 (1H, m, H-1), 2.18 (2H, t, J)6.7 Hz, H-2′a,b), 2.00
(4H, H-8′a.b, H-11′a,b), 1.62 (2H, m, H-3′a,b), 1.30 (20H, m,
H-4′a,b to H-7′a,b, and H-12′a,b to H-17′a,b), 1.00 (1H, m, H-3a),
0.87 (3H, t, J)7.1 Hz, H-18′), 0.58 (1H, m., H-3b). 13C NMR
(125 MHz Varian INOVA, CDCl3): δ175.8 (C-1′, s), 130.2 (C-9′,
C-10′, d), 49.2 (C-2, d), 36.2 (C-2′, t), 32.1 (C-4′to C-7′, C-12′to
C-16′, t), 29.6 (C-3′, t), 27.8 (C-1, d), 26.1 (C-3, t), 24.7 (C-8′,
C-11′, t), 23.1 (C-17′, t), 14.3 (C-18′, q). ESI-MS: m/z360 [M +
Na]+.
(1R,2S)-N-Oleoylcyclopropanolamide (14b). Brown powder. [R]D
22
+4(c0.2, CHCl3). For 1H NMR, 13C NMR, and MS data, see
those of 13b.
3. Determination of the Absolute Configuration of the Cyclo-
propanolamides of Arachidonic and Oleic Acid. (a) Cyclopro-
panolamides of Arachidonic Acid (11a, 13a). To a solution of
compound 11a (1.5 mg) in dry pyridine (0.3 mL), an excess of
(R)- or (S)-MTPA chloride was added and the mixture left at room
temperature for 12 h under stirring. The reaction mixture was then
diluted with ether and washed with H2O and saturated NaCl aqueous
solution. The organic layer was dried over Na2SO4, concentrated
under reduced pressure to give (S)-MTPA ester (1.1 mg) and
R-MTPA ester (1.2 mg), respectively (Figure 4). (S)-MTPA ester
of 13a: colorless oil. 1H NMR (CDCl3, 500 MHz): δ7.53 (3H, m,
MTPA phenyl), 7.42 (2H, m, MTPA phenyl), 4.40 (1H, m, H-2),
3.61 (3H, s, MTPA-OMe), 3.08 (1H, m, H-1), 1.37 (1H, m, H-3a),
0.91 (1H, m, H-3b). The signals of the arachidonoyl portion are
identical to those of 13a.(R)-MTPA ester of 13a: colorless oil. 1H
NMR (CDCl3, 500 MHz): δ7.56 (3H, m, MTPA phenyl), 7.42
(2H, m, MTPA phenyl), 4.40 (1H, m, H-2), 3.56 (3H, s, MTPA-
OMe), 3.05 (1H, m, H-1), 1.45 (1H, m, H-3a), 0.96 (1H, m, H-3b).
The signals of the arachidonoyl portion are identical to those of
13a. To a solution of compound 11a (1.2 mg) in dry pyridine (0.3
mL), an excess of (R)- or (S)-MTPA chloride was added and treated
as described above, affording (S)-MTPA ester (1.0 mg) and
R-MTPA (1.0 mg) ester of 11a, respectively (Figure 4). (S)-MTPA
ester of 11a: colorless oil. 1H NMR (CDCl3, 500 MHz): δ7.53
(3H, m, MTPA phenyl), 7.42 (2H, m, MTPA phenyl), 4.37 (1H,
m, H-2), 3.61 (3H, s, MTPA-OMe), 2.96 (1H, m, H-1), 1.37 (1H,
m, H-3a), 1.15 (1H, m, H-3b). The signals of the arachidonoyl
portion are identical to those of 11a.(R)-MTPA ester of 11a:
colorless oil. 1H NMR (CDCl3, 500 MHz): δ7.56 (3H, m, MTPA
phenyl), 7.42 (2H, m, MTPA phenyl), 4.37 (1H, m, H-2), 3.56 (3H,
s, MTPA-OMe), 2.90 (1H, m, H-1), 1.39 (1H, m, H-3a), 1.17 (1H,
H-3b). The signals of the arachidonoyl portion identical to those
of 11a.
(b) Cyclopropanolamides of Oleic Acid (11b, 13b). To a solution
of compound 11b (2.0 mg) in dry pyridine (0.5 mL), an excess of
(R)- or (S)-MTPA chloride was added and treated as described
above, affording the (S)-MTPA ester (1.6 mg) and R-MTPA (1.6
mg) esters of 11b, respectively (Figure 4). (S)-MTPA ester of 11b:
amorphous solid. 1H NMR (CDCl3, 500 MHz): δ7.53 (3H, m,
MTPA phenyl), 7.42 (2H, m, MTPA phenyl), 4.37 (1H, m, H-2),
3.61 (3H, s, MTPA-OMe), 3.11 (1H, m, H-1), 1.39 (1H, m, H-3a),
0.89 (1H, m, H-3b). The signals of the oleoyl moiety are identical
to those of 11b.(R)-MTPA ester of 11b: amorphous solid. 1H NMR
(CDCl3, 500 MHz): δ7.56 (3H, m, MTPA phenyl), 7.42 (2H, m,
MTPA phenyl), 4.37 (1H, m, H-2), 3.56 (3H, s, MTPA-OMe), 3.08
(1H, m, H-1), 1.44 (1H, m, H-3a), 0.91 (1H, m, H-3b). The signals
of the oleoyl moiety are identical to those of 11b.
To a solution of compound 13b (1.6 mg) in dry pyridine (0.4
mL), an excess of (R)- or (S)-MTPA chloride was added, and the
solution was treated as above, affording (S)-MTPA ester (1.2 mg)
and R-MTPA ester (1.2 mg) of 13b, respectively (Figure 4). (S)-
MTPA ester of 13b: amorphous solid. 1H NMR (CDCl3, 500 MHz):
δ7.53 (3H, m, MTPA phenyl), 7.42 (2H, m, MTPA phenyl), 4.40
(1H, m, H-2), 3.61 (3H, s, MTPA-OMe), 2.92 (1H, m, H-1), 1.37
(1H, m, H-3a), 1.10 (1H, m, H-3b). The signals of the oleoyl moiety
are identical to those of 13b.(R)-MTPA ester of 13b: amorphous
solid. 1H NMR (CDCl3, 500 MHz): δH7.56 (3H, MTPA phenyl,
m), 7.42 (2H, MTPA phenyl, m), 4.40 (1H, H-2, m), 3.56 (3H,
MTPA-OMe, s), 2.86 (1H, H-1, m), 1.40 (1H, H-3a, m), 1.16 (1H,
H-3b, m). The signals of the oleoyl moiety were identical to those
of 13b.
4. Cannabinoid CB1and CB2Receptor Binding Assays. Mem-
branes from HEK-293 cells transfected with the human recombinant
CB1receptor (Bmax )2.5 pmol/mg protein) and human recombinant
CB2receptor (Bmax )4.7 pmol/mg protein) were incubated with
[3H]CP-55,940 (0.14 nM, Kd)0.18 and 0.084 nM, Kd)0.31
nM, respectively, for CB1and CB2receptors) as the high affinity
ligand and displaced with 10 µM WIN 55212-2 as the heterologous
competitor for nonspecific binding (Kivalues of 9.2 and 2.1 nM,
respectively, for CB1and CB2receptor). All compounds were tested
following the procedure described by the manufacturer (Perkin-
Fatty Acid Ethanolamides as Probes Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3007

Elmer, Italy). Displacement curves were generated by incubating
drugs with [3H]CP-55,940 for 90 min at 30 °C. Kivalues were
calculated by applying the Cheng-Prusoff equation to the IC50
values (obtained by GraphPad) for the displacement of the bound
radioligand by increasing concentrations of the test compound. Data
are reported as mean values (SEM of at least n)3 experiments.
5. Cannabinoid CB1Receptor Functional Assay. The method
used for measuring agonist-stimulated [35S]GTPγS-binding to CB1
receptors was as described previously.
38
The assays were carried
out with mouse brain membranes (10 µgmL
-1), preincubated for
30 min at 30 °C with 0.5 U mL-1adenosine deaminase (200 U
mg-1) to remove endogenous adenosine, GTPγS binding buffer (50
mM Tris-HCl, 50 mM Tris-Base, 5 mM MgCl2, 1 mM EDTA, 100
mM NaCl, 1 mM dithiothreitol, 0.1% BSA) in the presence of 0.1
nM [35S]GTPγS and 30 µM GDP, in a final volume of 500 µL.
Binding was initiated by the addition of [35S]GTPγS to the wells.
Nonspecific binding was measured in the presence of 30 µM
GTPγS. The compounds were incubated in the assay for 60 min at
30 °C. The mixture was terminated by a rapid vacuum filtration
method using a washing buffer (50 mM Tris-HCl, 50 mM Tris-
Base, 0.1% BSA), and the radioactivity was quantified by liquid
scintillation spectrometry. Compounds were stored as a stock
solution of 10 mM in DMSO, the vehicle concentration in all assay
wells being 0.1% DMSO. Net agonist stimulated [35S]GTPγS-
binding values were calculated by subtracting basal binding values
(obtained in the absence of agonist) from agonist-stimulated values
(obtained in the presence of agonist) as detailed elsewhere.
39
Values
for EC50 and maximal effect (Emax) have been calculated by
nonlinear regression analysis using the equation for a sigmoid
concentration-response curve (GraphPad Prism5.0).
6. TRPV1 Channel Activity Assay. HEK-293 cells stably
overexpressing recombinant human TRPV1 cDNA were grown as
monolayers in minimum essential medium supplemented with
nonessential amino acids, 10% fetal calf serum, and 2 mM
glutamine and maintained under O2/CO2(95%/5%) at 37 °C. The
effect of the substances on Ca2+influx was determined by using
Fluo-4, a selective intracellular fluorescent probe for Ca2+.Onthe
day of the experiment, the cells (50-60000 per well) were loaded
for1hat25°C with Fluo-4 methyl ester (Molecular Probes,
Invitrogen), 4 µM in dimethyl sulfoxide containing 0.02% Pluronic
(Molecular Probes, Invitrogen), in minimum essential medium
without fetal bovine serum. After being loaded, cells were washed
twice in Tyrode’s buffer, pH 7.4 (NaCl 145 mM, KCl 2.5 mM,
CaCl21.5 mM, MgCl21.2 mM, D-glucose 10 mM, HEPES 10 mM,
pH 7.4), resuspended in Tyrode’s buffer, and transferred (50-60000
cells) to the quartz cuvette of the fluorescence detector (Perkin-
Elmer LS50B) under continuous stirring. Experiments were carried
out by measuring cell fluorescence at 25 °C(λEX )488 nm,
λEM )516 nm) before and after the addition of the test compounds
at various concentrations. Agonist activity was determined in
comparison to the maximum Ca2+influx due to the application of
4µM ionomycin (Sigma). EC50 values were determined as the
concentration of test substances required to produce half-maximal
increases in [Ca2+]i. All determinations were at least performed in
triplicate. Curve fitting (sigmoidal dose-response variable slope)
and parameter estimation were performed with GraphPad Prism
(GraphPad Software Inc., San Diego, CA).
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