New tetrazole-based selective anandamide uptake inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.04.003

A new series of 1,5- and 2,5-disubstituted tetrazoles have been synthesized and evaluated as inhibitors of anandamide cellular uptake. Some of them inhibit the uptake process with a relatively high potency (IC₅₀ = 2.3-5.1 μM) and selectively over other proteins involved in endocannabinoid action and metabolism.

Full text of DOI:10.1016/j.bmcl.2008.04.003

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2820–2824
New tetrazole-based selective anandamide uptake inhibitors
Giorgio Ortar,a,* Aniello Schiano Moriello,b Maria Grazia Cascio,b
Luciano De Petrocellis,c Alessia Ligresti,b Enrico Morera,a Marianna Nallia and
Vincenzo Di Marzob
a
`
Dipartimento di Studi Farmaceutici, ‘Sapienza’ Universita di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy
bIstituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
cIstituto di Cibernetica, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
Received 13 February 2008; revised 31 March 2008; accepted 1 April 2008
Available online 4 April 2008
Abstract—A new series of 1,5- and 2,5-disubstituted tetrazoles have been synthesized and evaluated as inhibitors of anandamide
cellular uptake. Some of them inhibit the uptake process with a relatively high potency (IC50 = 2.3–5.1 lM) and selectively over
other proteins involved in endocannabinoid action and metabolism.
Ó 2008 Elsevier Ltd. All rights reserved.
The endocannabinoid signalling system is composed of
two G-protein coupled receptors, CB1 and CB2, their
endogenous agonists (endocannabinoids), and a series
of proteins responsible for the synthesis and the inacti-
vation of endocannabinoids.1 This system has been
found to be involved in an increasing number of physi-
ological and pathological conditions, and an enormous
interest has therefore arisen about the potential thera-
peutic applications of compounds acting on its compo-
nents.2 Endocannabinoids are synthesized and released
‘on demand’, act near their site of synthesis, and are
then rapidly inactivated by cellular uptake followed by
intracellular hydrolysis by specific enzymes. Inhibitors
of endocannabinoid degradation could offer a rational
therapeutic approach to a variety of diseases including
pain, anxiety, cancer and neurodegenerative disorders
in which the elevation of endocannabinoid levels repre-
sent an adaptive reaction to re-establish normal homeo-
stasis when this is pathologically perturbed.3
(FAAH),6 whereas a monoacylglycerol lipase (MAGL)7
is critical in degrading 2-AG.
To date, the existence of an AEA membrane transporter
independent of FAAH activity remains, however, ques-
tionable and alternative hypotheses have been proposed,
including passive diffusion, endocytosis and intracellular
sequestration.8 While the nature of the mechanism
responsible for the AEA uptake is still a matter of de-
bate, the recent individuation of a number of ananda-
mide-based selective AEA transport inhibitors that do
not appear to inhibit FAAH has stimulated further
studies to distinguish between the relative contributions
of the putative AEA transporter and FAAH in AEA re-
moval.8d,9 From a medicinal chemistry perspective, the
identification of potent and selective inhibitors of AEA
transport lacking a long acyl side chain would be advan-
tageous in terms of more precise target identification
and of drug-like characteristics.
The levels of the two most studied endocannabinoids,
anandamide (AEA)4 and 2-arachidonoylglycerol (2-
AG),5 appear to be regulated in different, and sometimes
even opposing ways. AEA is assumed to be transported
into the cell by a specific transporter and then rapidly
hydrolyzed by the enzyme fatty acid amide hydrolase
In this respect, Hopkins and Wang reported in 2004 in
abstract form a non-arachidonoyl compound, SEP-
0200228, that inhibited AEA uptake in human mono-
cytes.10 In 2006, Moore et al. described a potent, com-
petitive carbamoyl tetrazole inhibitor of AEA uptake,
LY2318912, which has allowed the identification of a
high-affinity AEA binding site distinct from FAAH in
RBL-2H3 cell plasma membranes.11 However, the same
group subsequently reported that both LY2183240 (1)
(Fig. 1), the parent compound of LY2318912, and a
N-cyclopropyl analogue of SEP-020022812 do inhibit
Keywords: Endocannabinoids; Anandamide uptake; Anandamide up-
take inhibitors; Tetrazoles.
*
Corresponding author. Tel.: +39 6 49913612; fax: +39 6 491491;
e-mail: giorgio.ortar@uniroma1.it
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.003

G. Ortar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2820–2824
2821
The effects of tetrazoles 6–15 on (a) [14C]anandamide
hydrolysis by rat brain membranes (which express
FAAH as the only AEA hydrolyzing enzyme); (b)
[14C]anandamide uptake by intact RBL-2H3 cells
(where a putative AEA transporter has been character-
ized pharmacologically); (c) [14C]2-AG hydrolysis by
COS cell cytosolic fractions (which express MAGL);
(d) sn-1-[14C]oleoyl-2-arachidonoyl-glycerol (DAG)
conversion into 2-AG and [14C]oleic acid by COS cells
membranes overexpressing the human recombinant
DAGLa; (e) CB1 and CB2 receptors in transfected hu-
man COS cells; and (f) intracellular Ca2+ elevation med-
iated by transient receptor potential ankyrin type 1
(TRPA1) and vanilloid type 1 (TRPV1) channels over-
expressed in HEK-293 cells, are shown in Table 1.20
The effects on these two channels were studied since
TRPV1, like cannabinoid CB1 and CB2 receptors, is
activated by AEA and can be activated indirectly also
following FAAH inhibition and the subsequent eleva-
tion of AEA levels,21 whereas TRPA1 has been reported
to be activated by the widely used FAAH inhibitor
URB-597.22
O
N
N
N
N
N
O
N
N
N
N
N
1
2
(LY2183240)
O
O
O
N
N
O
R
N
H
N
N
N
R
N
N
H
N
N
N
N
3
4
: R = 3-biphenyl
5
: R = 4-biphenyl
: R = Ph(CH2)6
Figure 1. Structures of carbamoyl tetrazoles previously described as
non-selective inhibitors of AEA cellular uptake.
enzyme activity in purified FAAH preparations,13 and
Alexander and Cravatt eventually demonstrated that 1
(actually a mixture of 1 and its 2,5-regioisomer 2)14 is
a potent, covalent inhibitor not only of FAAH but also
of several other serine proteases, including MAGL, via
carbamylation of an active site catalytic serine.15
None of the compounds inhibited significantly FAAH,
MAGL and DAGL. Thus, the substitution of the carb-
amylating N,N-dimethylaminocarbonyl group proved to
be detrimental to the inhibitory potency on the three ser-
ine hydrolases, despite the fact that some of them could
still have acted by way of their electrophilic group inter-
acting with the catalytic triad to produce a covalent
intermediate, as assumed for a-ketoheterocycle-based
inhibitors23 and some bis-arylimidazole esters.24 How-
ever, compounds 6a, 7a, 9b, 10a and 11b retained the
ability to inhibit efficaciously the uptake process with
IC50 values in the low micromolar range (2.3–5.1 lM),
a result which seems to represent yet another piece of
evidence in favour of the existence of a FAAH-indepen-
dent mechanism for AEA removal from the extracellular
milieu. Consistent with the previous observations,14 1,5-
isomers are generally significantly more potent inhibi-
tors than the corresponding 2,5 counterparts (9b repre-
senting the only exception to this rule). The most
potent inhibitor (11b), however, belongs to the latter
class of regioisomers. Unfortunately, as mentioned be-
fore, the 1,5-regioisomer 11a, which might have been
even more potent, could not be evaluated since it was
formed in a very low yield and could not be isolated
in a pure form. The inhibitory activity seems to depend
on the presence of a functional group that might con-
ceivably reinforce the interaction with the protein(s) in-
volved in membrane transport, as compounds lacking
any functionality at N1 (8a, b and 13a, b) were unable
to inhibit AEA uptake process. Overall, the putative
AEA membrane transporter appears to display a weak
preference for the 4-biphenyl over the biphenyl-4-car-
boxamido moieties. All compounds exhibited weak or
no affinity for CB1, CB2, TRPA1, and TRPV1 receptors.
In particular, of the five most active compounds on
AEA uptake, only one (9b) exhibited measurable activ-
ity at CB2 and TRPA1 receptors. When at all active
on cannabinoid receptors, the new compounds preferred
CB2 over CB1 receptors, with only one exception (8b).
Opposite to what observed for AEA uptake, the 2,5-iso-
In order to gain further insight into the actions of the
carbamoyl tetrazole class of compounds, we embarked
recently on a structure-activity relationship (SAR) study
focused on the nature of the 5-substitution and of the
regiochemistry.14 Five of the 18 compounds synthesized
and evaluated for their activity on AEA uptake, FAAH,
MAGL, and DAGL (diacylglycerol lipase catalyzing the
biosynthesis of 2-AG)16 were found to be active on AEA
uptake, although all potently inhibited FAAH and some
of them MAGL and DAGL as well (compounds 1–5)
(Fig. 1). With the aim of ‘designing out’ these cross-
activities and attain a good selectivity towards the
AEA uptake process, we have now investigated the ef-
fect of replacing the N,N-dimethylaminocarbonyl por-
tion of previous tetrazolic ureas with analogous groups
devoid of inherent carbamylating reactivity. We report
here that this modification indeed results in the identifica-
tion of some new tetrazole-based selective AEA uptake
inhibitors.
From the set of compounds 1–5, the most promising
4-biphenyl and biphenyl-4-carboxamido moieties were
selected, since 1 and 5 were already lacking inhibitory
activity against MAGL and DAGL. The tetrazoles listed
in Table 1 were prepared by the alkylation of 5-[(biphenyl-
4-yl)methyl]-1H-tetrazole (16)14 or of N-[(1H-tetrazol-5-
yl)methyl]biphenyl-4-carboxamide (17)14 (Scheme 1).17
From the mixture of regioisomers 11a + 11b, only 2,5-reg-
ioisomer 11b could be isolated in a pure form. The struc-
tures of the regioisomeric tetrazoles were assigned on the
basis of their 1H and 13C NMR spectra.18 NMR studies of
disubstituted tetrazoles, included those of our previous
paper, have in fact shown that protons of the CH2 group
attached to N1 and CN4 carbon atom of 1,5-disubstituted
tetrazoles are more shielded than the corresponding
atoms of the 2,5-disubstituted tetrazoles.19

Table 1. Effect of tetrazoles 6–15 on [14C]anandamide hydrolysis by rat brain membranes, [14C]anandamide uptake by intact RBL-2H3 cells, [14C]2-AG hydrolysis by COS cell cytosolic fractions, sn-1-
14C]oleoyl-2-arachidonoyl-glycerol (DAG) conversion into 2-AG and [14C]oleic acid by COS cells membranes, cannabinoid CB1 and CB2 receptors in transfected human COS cells, and transient receptor
potential TRPA1 and TRPV1 receptors transfected in HEK-293 cellsa
[
R2
N
N
N
R1
R1
R2
N
N
N
N
N
a
b
: R1 = 4-biphenyl-4-carboxamido; R2 = CH2CON(CH3)2
: R1 = 4-biphenyl-4-carboxamido; R2 = CH2COCH3
: R1 = 4-biphenyl; R2 = CH2CON(CH3)2
: R1 = 4-biphenyl; R2 = CH2COCH3
11
12
6
7
13: R1 = 4-biphenyl-4-carboxamido; R2 = CH2CH(CH3)2
14: R1 = 4-biphenyl-4-carboxamido; R2 = CH2CN
15: R1 = 4-biphenyl-4-carboxamido; R2 = CH2CO2CH3
8: R1 = 4-biphenyl; R2 = CH2CH(CH3)2
9: R1 = 4-biphenyl; R2 = CH2CN
10: R1 = 4-biphenyl; R2 = CH2CO2CH3
Compound
AEA hydrolysis
(IC50, lM)
AEA uptake
(IC50, lM)
2-AG hydrolysis
(IC50, lM)
DAG hydrolysis to 2-AG
(IC50, lM)
CB1 (Ki, lM)
CB2 (Ki, lM)
TRPA1
(EC50, lM)
TRPA1
(efficacy)b
TRPV1
(EC50, lM)
TRPV1
(efficacy)c
6a
6b
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.6
>10
>10
>10
5.2
>100
>100
>100
>100
>100
26.3
0
0
>100
>100
>100
>100
>100
10.0
4.0
5.3
0
>25
5.0
7a
7b
11.4
61.4
50.8
136.0
83.2
101.7
0
>25
>25
>25
24.8
5.1
0
8a
8b
7.7
4.7
4.7
20.7
5.4
0
9a
9b
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6.2
26.3
16.2
>100
>100
>100
>100
>100
>100
>100
15.0
10a
10b
11b
12a
12b
13a
13b
14a
14b
15a
15b
4.9
>10
8.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0
>25
2.3
36.0
0
0
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.2
7.7
0
12.5
>25
>25
>25
>25
>25
>25
>25
0
14.0
26.1
0
9.1
10.2
6.3
2.6
8.6
2.2
53.5
0
>100
>100
29.6
0
0
0
>100
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 Expressed the effect of a 100 lM concentration of each compound as percent of the effect of 100 lM mustard oil.
c Expressed as the effect of a 10 lM concentration of each compound as percent of the effect on intracellular Ca2+ of 4 lM ionomycin.

G. Ortar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2820–2824
2823
R2 Cl, DIPEA, CH2Cl2 (or THF)
6a 6b
7a 7b 9a 12a 9b 12b
or
+
(or
14a 15a 14b 15b
)
+
-
+
-
or
,
+
,
H
rt (or 60 °C), 15 h
N
N
R1
N
N
(CH3)2CHCH2Cl, Cs2CO3, DMF
80 °C, 15 h
: R = biphenyl-4-carboxamido
8a 8b
13a 13b
+ )
+
(or
1
16
17
: R = 4-biphenyl
1
R2 = CH2CON(CH3)2,, CH2COCH3, CH2CN, CH2CO2CH3
Scheme 1. Synthesis of tetrazoles 6–15.
6. (a) Bracey, M. H.; Hanson, M. A.; Masuda, K. R.;
Stevens, R. C.; Cravatt, B. F. Science 2002, 298, 1793; (b)
Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.;
Lerner, R. A.; Gilula, N. B. Nature 1996, 384, 83; (c)
Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 2238; (d) Patricelli, M. P.; Cravatt, B. F. Vitam.
Horm. 2001, 62, 95.
7. Dinh, T. P.; Carpenter, D.; Leslie, F. M.; Freund, T. F.;
Katona, I.; Sensi, S. L.; Kathuria, S.; Piomelli, D. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 10819.
8. (a) Glaser, S. T.; Kaczocha, M.; Deutsch, D. G. Life Sci.
2005, 77, 1584; (b) Oddi, S.; Bari, M.; Battista, N.;
Barsacchi, D.; Cozzani, I.; Maccarrone, M. Cell. Mol. Life
Sci. 2005, 62, 386; (c) Ligresti, A.; Morera, E.; Van Der
Stelt, M.; Monory, K.; Lutz, B.; Ortar, G.; Di Marzo, V.
Biochem. J. 2004, 380, 265; (d) Fegley, D.; Kathuria, S.;
Mercier, R.; Li, C.; Goutopoulos, A.; Makriyannis, A.;
Piomelli, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8756;
(e) McFarland, M. J.; Barker, E. L. Pharmacol. Ther.
2004, 104, 117; (f) Glaser, S. T.; Abumrad, N. A.; Fatade,
F.; Kaczocha, M.; Studholme, K. M.; Deutsch, D. G.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4269; (g) Hillard,
C. J.; Jarrahian, A. Br. J. Pharmacol. 2003, 140, 802.
mers were more potent than the 1,5 isomers on both CB2
and TRPA1. All compounds exhibited very weak, and
often hardly measurable, efficacy at TRPV1 receptors,
and therefore the EC50 values calculated for this type
of activity have little pharmacological meaning. Interest-
ingly, compounds 8b, 9a and 9b were as efficacious as
the
mustard
oil
(allyl
isothiocyanate)
(EC50 = 2.5 0.7 lM, efficacy 100%) on TRPA1-medi-
ated increase of intracellular Ca2+, albeit less potent.
This suggests that, like previously shown for the car-
bamoyl-derivative, URB-597,22 tetrazole-containing
derivatives also can interact with this ion channel, which
has been involved in pain transduction by peripheral
sensory neurons. Since these latter compounds, unlike
URB-597, lack the capability of covalently binding to
nucleophilic groups like serine hydroxyl groups or, in
the case of TRPA1, cysteine sulphydryl groups, this
finding also supports the concept that not all TRPA1
agonists possess sulphydryl reacting moieties in their
chemical structure.25
´
9. (a) Lopez-Rodrıguez, M. L.; Viso, A.; Ortega-Gutierrez,
S.; Fowler, C. J.; Tiger, G.; de Lago, E.; Fernandez-Ruiz,
´
´
In conclusion, we have shown here that it is possible to
develop, through appropriate modifications of the com-
pounds introduced by Eli Lilly and subsequently studied
and extended by us, selective and non-fatty acid-based
AEA uptake inhibitors that might be useful in the eluci-
dation of the ‘vexed question of the AEA transporter
protein’.1
´
J.; Ramos, J. A. J. Med. Chem. 2003, 46, 1512; (b) Ortar,
G.; Ligresti, A.; De Petrocellis, L.; Morera, E.; Di Marzo,
V. Biochem. Pharmacol. 2003, 65, 1473.
10. Hopkins, S. C.; Wang, F. Symposium on the Canabinoids;
International Cannabinoid Research Society: Burlington,
VT, 2004, p. 103.
11. Moore, S. A.; Nomikos, G. G.; Dickason-Chesterfield, A.
K.; Schober, D. A.; Schaus, J. M.; Ying, B.-P.; Xu, Y.-C.;
Phebus, L.; Simmons, R. M. A.; Li, D.; Iyengar, S.;
Felder, C. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
17852.
12. Aquila, B. A.; Hopkins, S.; Lockshin, C. A.; Wang, F.
WO Patent 03097573, 2003.
13. Dickason-Chesterfield, A. K.; Kidd, S. R.; Moore, S. A.;
Schaus, J. M.; Liu, B.; Nomikos, G. G.; Felder, C. C. Cell.
Mol. Neurobiol. 2006, 26, 407.
References and notes
1. (a) Alexander, S. P. H.; Kendall, D. A. Br. J. Pharmacol.
2007, 152, 602; (b) Lambert, D. M.; Fowler, C. J. J. Med.
Chem. 2005, 48, 5059; (c) Howlett, A. C. Prostaglandins
Other Lipid Mediat. 2002, 68, 619; (d) Sugiura, T.; Waku,
K. J. Biochem. 2002, 132, 7.
2. (a) Di Marzo, V.; De Petrocellis, L. Ann. Rev. Med. 2006,
57, 553; (b) Jonsson, K. O.; Holt, S.; Fowler, C. J. Basic
Clin. Pharmacol. Toxicol. 2006, 98, 124; (c) Mackie, K.
Ann. Rev. Pharmacol. Toxicol. 2006, 46, 101.
14. Ortar, G.; Cascio, M. G.; Schiano Moriello, A.; Camalli,
M.; Morera, E.; Nalli, M.; Di Marzo, V. Eur. J. Med.
Chem. 2008, 43, 62.
15. Alexander, J. P.; Cravatt, B. F. J. Am. Chem. Soc. 2006,
128, 9699.
3. Di Marzo, V.; Petrosino, S. Curr. Opin. Lipidol. 2007, 18,
129.
16. Di Marzo, V.; Bifulco, M.; De Petrocellis, L. Nat. Rev.
Drug Discov. 2004, 3, 771.
4. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.;
Stevenson, L. A.; Griffin, G.; Gibson, D.; Mandelbaum,
A.; Etinger, A.; Mechoulam, R. Science 1992, 258, 1946.
5. (a) Mechoulam, R.; Ben-Shabat, S.; Hanus, L.; Ligumsky,
M.; Kaminski, N. E.; Schatz, A. R.; Gopher, A.; Almog,
S.; Martin, B. R.; Compton, D. R.; Pertwee, R. G.;
Griffin, G.; Bayewitch, M.; Barg, J.; Vogel, Z. Biochem.
Pharmacol. 1995, 50, 83; (b) Sugiura, T.; Kondo, S.;
Sukagawa, A.; Nakane, S.; Shinoda, A.; Itoh, K. Biochem.
Biophys. Res. Commun. 1995, 215, 89.
17. General procedure for the synthesis of compounds 6–15.
To a stirred solution of tetrazole 1614 or 1714 and N,N-
diisopropylethylamine (3–6 equiv) in CH2Cl2 (3 mL/mmol
of tetrazole), the appropriate chloro derivative (1.5–3
equiv) was added and the mixture was stirred at room
temperature overnight (in the case of compounds 14, the
reaction was carried out at 60 °C in THF, while in the case
of compounds 8 and 13 the reaction was run at 80 °C in

2824
G. Ortar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2820–2824
DMF with Cs2CO3 as the base). The mixture was diluted
128.96, 131.90, 138.96, 143.18, 153.98, 166.34, 199.80.
Anal. (C18H17N5O2) C, H, N.
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 on silica gel using hexane/AcOEt
or CH2Cl2/AcOEt mixtures as eluents, the less polar 2,5-
disubstituted tetrazole (16–70% yields) eluting prior to the
1,5-disubstituted regioisomer (14–53% yields).
19. (a) May, B. C. H.; Abell, A. D. Tetrahedron Lett.
2001, 42, 5641; (b) Bethel, P. A.; Hill, M. S.; Mahon,
M. F.; Molloy, K. C. J. Chem. Soc. Perkin Trans. 1
1999, 3507; (c) Byard, S. J.; Herbert, J. M. Tetrahe-
dron 1999, 55, 5931.
20. Detailed procedures for assays of AEA cellular uptake by
rat basophilic RBL-2H3 cells, and of rat FAAH, human
DAGL, MAGL, CB1, CB2, and TRPV1 activities were
reported (see Ref. 14 and: Di Marzo, V.; Ligresti, A.;
Morera, E.; Nalli, M.; Ortar, G. Bioorg. Med. Chem. 2004,
12, 5161). Assays for activity at TRPA1 channels were
carried out as follows. HEK-293 cells stably transfected
with the cDNA encoding for the rat TRPA1 receptor were
plated on 10 cm diameter Petri dishes and after 3 days
were loaded for 1 h at room temperature with the
cytoplasmic calcium indicator Fluo4-AM 4 lM (Molecu-
lar Probes) containing Pluronic (0.02%, Molecular
Probes). Cells were washed twice in Tyrode’s buffer pH
7.4 (NaCl 145 mM; KCl 2.5 mM; CaCl2 1.5 mM; MgCl2
1.2 mM; D-Glucose 10 mM; HEPES 10 mM pH 7.4),
resuspended in Tyrode’s buffer and transferred to the
quartz cuvette of the spectrofluorimeter (kex = 488 nm;
18. Data for selected compounds: Compound 6a: yield 53%;
mp 181–182 °C (from CHCl3); IR (CHCl3) 3034, 2932,
1675, 1602, 1488, 1406, 1264, 1150 cmÀ1 1H NMR
;
(300 MHz, CDCl3) d 2.95 (3H, s), 2.99 (3H, s), 4.40 (2H,
s), 4.90 (2H, s), 7.23–7.58 (9H, m); 13C NMR (75 MHz,
CDCl3) d 29.54, 36.00, 36.52, 48.39, 126.99, 127.64,
127.72, 128.92, 129.31, 132.72, 140.23, 140.75, 154.89,
163.50. Anal. (C18H19N5O) C, H, N.
Compound 7a: yield 23%; mp 145–146 °C (from hexane/
CH2Cl2); IR (KBr) 3031, 2974, 1731, 1517, 1484, 1459,
1
1445, 1352, 1250, 1176, 1128 cmÀ1; H NMR (300 MHz,
CDCl3) d 2.13 (3H, s), 4.30 (2H, s), 4.94 (2H, s), 7.18–7.58
(9H, m); 13C NMR (75 MHz, CDCl3) d 26.98, 29.40, 55.80,
127.00, 127.71, 127.91, 128.92, 129.13, 132.23, 140.07,
140.98, 154.52, 197.24. Anal. (C17H16N4O) C, H, N.
Compound 9b: yield 46%; mp 86–87 °C (from hexane/
CH2Cl2); IR (KBr) 3033, 2997, 2360, 1487, 1428, 1407,
k
em = 516 nm). (Perkin-Elmer LS50B) under continuous
1
1366, 1204, 1148, 1074, 1016 cmÀ1; H NMR (300 MHz,
stirring. Intracellular Ca2+ concentration ([Ca2+]i) was
determined before and after the addition of various
concentrations of test compounds. Potency was expressed
as the concentration exerting a half-maximal agonist effect
(i.e. half-maximal increases in [Ca2+]i) (EC50), calculated
by using GraphPadÒ. The efficacy of the agonists was
determined by normalizing their effect to the maximal
effect on [Ca2+]i observed with 100 lM mustard oil.
CDCl3) d 4.31 (2H, s), 5.46 (2H, s), 7.32–7.58 (9H, m); 13
C
NMR (75 MHz, CDCl3) d 31.49, 39.80, 111.18, 127.06,
127.36, 127.56, 128.79, 129.31, 134.83, 140.26, 140.63,
167.16. Anal. (C16H13N5) C, H, N.
Compound 10a: yield 51%; mp 115 °C (from hexane/
CH2Cl2); IR (KBr) 3004, 2958, 1737, 1513, 1488, 1438,
1408, 1230, 1114 cmÀ1; 1H NMR (300 MHz, CDCl3) d 3.67
(3H, s), 4.35 (2H, s), 4.95 (2H, s), 7.22–7.57 (9H, m); 13C
NMR (75 MHz, CDCl3) d 29.26, 47.94, 53.18, 126.98,
127.64, 127.84, 128.89, 129.12, 132.09, 140.16, 140.89,
154.42, 165.46. Anal. (C17H16N4O2) C, H, N.
Compound 11b: yield 36%; mp 185–187 °C (from MeOH);
IR (KBr) 3319, 3054, 2932, 1664, 1646, 1609, 1540, 1506,
1485, 1407, 1307, 1155 cmÀ1; 1H NMR (300 MHz, DMSO-
d6) d 2.72 (3H, s), 3.05 (3H, s), 4.77 (2H, d, J = 5.6 Hz), 5.65
(2H, s), 7.40–7.98 (9H, m), 9.29 (1H, m); 13C NMR
(75 MHz, DMSO-d6) d 32.16, 35.10, 35.77, 48.36, 126.49,
126.81, 127.99, 128.09, 128.99, 131.93, 138.95, 143.13,
154.22, 164.38, 166.17. Anal. (C19H20N6O2) C, H, N.
Compound 12a: yield 30%; mp 196–198 °C (from MeOH);
IR (KBr) 3306, 2947, 1728, 1640, 1542, 1485,1462, 1416,
1324, 1180, 1129 cmÀ1; 1H NMR (300 MHz, DMSO-d6) d
2.27 (3H, s), 4.72 (2H, d, J = 5.6 Hz), 5.75 (2H, s), 7.40–
7.97 (9H, m), 9.32 (1H, m); 13C NMR (75 MHz, DMSO-
d6) d 26.82, 31.89, 55.78, 126.53, 126.80, 127.96, 128.06,
21. De Petrocellis, L.; Bisogno, T.; Maccarrone, M.; Davis, J.
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