7804 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Sinning et al.
stirred under ice cooling for 10 min. Then 50.8 mg (0.4 mmol)
oxalyl chloride was added, and the mixture was stirred under ice
cooling for 45 min. After the addition of 246.3 mg (2 mmol) 4, the
reagents were allowed to react 30 min at room temperature. The
reaction mixture was diluted with 25 mL TBME, decanted into a
round-bottom flask, and the solvent was removed by rotary
evaporation. The crude product was purified by column chroma-
tography on silica gel (40-63 µm) using TBME + 3% NEt3 as
FAAH Assay. The effect of the compounds on the enzymatic
hydrolysis of [14C]anandamide was studied by using membranes
prepared from rat brain. In brief, a half-rat brain was homogenized
in Tris-HCl (50 mM, pH 7.0) by using an Ultraturrax (16000 rpm,
3 min) and a Dounce homogenizer at 4 °C. Homogenates were
first centrifuged at 3000 rpm (4 °C, 6 min) to rid the debris, and
the supernatant was centrifuged at 11000 rpm (4 °C, 25 min). The
pellet from this latter centrifugation was resuspended in Tris-HCl
(50 mM, pH 9.0) and used for the assay. Tissue suspensions
(containing 70 µg of protein) were incubated with two concentra-
tions of the test compounds (10 and 50 µM) and [14C]anandamide
(10000 cpm) in Tris-HCl (50 mM, pH 9) for 30 min at 37 °C.
[14C]Ethanolamine produced from [14C]anandamide hydrolysis was
used to calculate FAAH activity and was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with 2 volumes of CHCl3/MeOH (1:1 by volume). All data
points were determined in duplicate.
Whole Blood Assay for COX-1 Inhibition. Blood was drawn
from a healthy 50 year old volunteer who had not taken any
nonsteroidal anti-inflammatory drug 2 weeks prior to blood
sampling. One mL aliquots of whole blood were immediately
transferred to plastic tubes containing 2 µL of the test agent
dissolved in DMSO. Blood was allowed to clot for 1 h at 37 °C.
Serum was separated by centrifugation, and TXB2 levels were
determined per GC-MS/MS as described previously.24 All data
points were determined in quadruplicate.
Whole Blood Assay for COX-2 Inhibition. One mL aliquots
of heparinized whole blood from a healthy 50 year old volunteer
who had not taken any nonsteroidal anti-inflammatory drug 2 weeks
prior to blood sampling were incubated with 10 µg of lipopolysac-
charides (from Escherichia coli 026:B26), 10 µg aspirin, plus 2
µL of the test agent dissolved in DMSO for 24 h at 37 °C.
Lipopolysaccharides were used as stimulants for COX-2 and the
contribution of platelet COX-1 activity was inhibited by aspirin.
Plasma was separated by centrifugation, and PGE2 levels were
determined per GC-MS/MS as described previously.24 All data
points were determined in quadruplicate.
Inhibition of Isolated Cyclooxygenases. A COX inhibitor
screening assay was used to determine the activity of isolated ovine
COX-1 and human recombinant COX-2 as described by the
manufacturer (Cayman Chemical Company, USA). Briefly, COX-1
or COX-2 was incubated with 5 at 37 °C for 10 min in a Tris-HCl
buffer (0.1 M, pH 8.0 containing 5 mM EDTA and 2 mM phenol).
Then, the COX reaction was initiated by addition of arachidonic
acid. The reaction was stopped 2 min later by 50 µL of 1 M HCl,
followed by addition of 100 µL saturated SnCl2 to reduce the COX-
produced PGH2 into PGF2R, which was further quantified by EIA
using PGE2 as standard. Blank was previously subtracted from each
value. Ketoprofen was used as a reference COX-inhibitor. Results
are means of four experiments.
1
eluent to afford 34.0 mg (20%) of a yellowish oil. H NMR (400
MHz, DMSO-d6), δ (ppm): 9.21 (s, 1H, OH), 8.12 (br s, 1H, CO-
3
4
NH), 7.01 (dd, J ) 6.6 Hz, J ) 2.1 Hz, 2H, 2 × Ar-H), 6.67
(dd, 3J ) 6.6 Hz, 4J ) 2.1 Hz, 2H, 2 × Ar-H), 5.39-5.27 (m, 8H,
3
4 × CHdCH), 4.11 (d, J ) 5.8 Hz, 2H, NH-CH2), 2.82-2.74
3
(m, 6H, 3 × CHdCH-CH2-CHdCH), 2.10 (t, J ) 7.5 Hz, 2H,
CH2-CH2-CO-NH), 2.05-1.98 (m, 4H, 2 × CHdCH-CH2), 1.55
(tt, 3J ) 3J ) 7.5 Hz, 2H, CH2-CH2-CO-NH), 1.33-1.20 (m, 6H,
CH2-CH2-CH2-CH3), 0.84 (t, 3J ) 6.8 Hz, 3H,... -CH3). 13C NMR
(100 MHz, CDCl3), δ (ppm): 173.1, 155.9, 130.5, 129.6, 129.2,
129.0, 128.9, 128.6, 128.3, 128.1, 127.9, 127.5, 115.7, 43.4, 36.2,
31.6, 29.4, 27.3, 26.7, 25.7, 25.6, 22.6, 14.1. EI-MS (70 eV), m/z
(%): 107 (100), 122 (28.6), 165 (21.4), 409 (3.6, M+). Anal.
(C27H39NO2) C, H, N.
N-(1H-Indazol-5-yl)acetamide (5). First, 266.4 mg (2 mmol)
5-aminoindazole were dissolved in 10 mL of dry THF and stirred
under ice cooling for 10 min. After the addition of 78.5 mg (1
mmol) acetyl chloride, the reagents were allowed to react 30 min
at room temperature. The reaction mixture was diluted with MeOH,
decanted into a round-bottom flask, and the solvent removed by
rotary evaporation. The crude product was purified by column
chromatography on silica gel (40-63 µm) using EtOAc/NEt3 (97/
3) as eluent. Crystallization from MeOH/TBME afforded 238.8 mg
(46%) of slightly violet crystals; mp: 203-206 °C (MeOH/TBME).
1H NMR (400 MHz, DMSO-d6), δ (ppm): 12.88 (br s, 1H, NH),
3
9.85 (s, 1H, CO-NH), 8.10-7.97 (m, 2H, 2 × Ar-H), 7.42 (d, J
3
4
) 8.7 Hz, 1H, Ar-H), 7.35 (dd, J ) 8.7 Hz, J ) 1.7 Hz, 1H,
Ar-H), 2.01 (s, 3H, CO-CH3). 13C NMR (100 MHz, CD3OD), δ
(ppm): 171.5, 138.9, 134.8, 133.0, 124.1, 122.9, 112.7, 111.1, 23.7.
EI-MS (70 eV), m/z (%): 133 (100), 175 (40, M+), 106 (20.7).
Anal. (C9H9N3O) C, H, N.
N-(4-Hydroxybenzyl)acetamide (6). First, 369.5 mg (3 mmol)
4 were dissolved in 10 mL of dry THF and stirred under ice cooling
for 10 min. After the addition of 117.8 mg (1.5 mmol) acetyl
chloride, the reagents were allowed to react for 30 min at room
temperature. The reaction mixture was diluted with MeOH, decanted
into a round-bottom flask, and the solvent was removed by rotary
evaporation. The crude product was purified by column chroma-
tography on silica gel (40-63 µm) using TBME/MeOH (95/5) as
eluent. Crystallization from EtOAc afforded 155.2 mg (63%) of
1
white crystals; mp: 124-126 °C (EtOAc)). H NMR (400 MHz,
DMSO-d6), δ (ppm): 9.22 (s, 1H, OH), 8.16 (br s, 1H, CO-NH),
3
4
3
7.03 (dd, J ) 6.2 Hz, J ) 2.1 Hz, 2H, 2 × Ar-H), 6.68 (dd, J
) 6.2 Hz, 4J ) 2.1 Hz, 2H, 2 × Ar-H), 4.10 (d, 3J ) 5.8 Hz, 2H,
NH-CH2), 1.82 (s, 3H, CO-CH3). 13C NMR (100 MHz, CD3OD),
δ (ppm): 172.7, 157.6, 130.5, 129.9, 116.1, 43.9, 22.6. EI-MS (70
eV), m/z (%): 165 (100, M+), 122 (72.1), 107 (47.9). Anal.
(C9H11NO2) C, H, N.
Biological Evaluation. CB1 and CB2 Assay. For CB1 and CB2
receptor binding assays, the compounds were tested using mem-
branes from HEK-293 cells overexpressing either the human
recombinant CB1 or CB2 receptor and [3H]CP-55,940 (Kd ) 0.18
nM for CB1 and 0.31 nM for CB2 receptors) as the high affinity
ligand as described by the manufacturer (Perkin-Elmer, Italy).
Membranes were incubated with increasing concentrations (1 nM
to 10 µM) of test compounds and [3H]CP-55,940 at 30 °C for 90
min, and the amount of displaced radioligand was determined by
scintillation counting. Ki values were calculated by applying the
Cheng-Prusoff equation to the IC50 values for the displacement
of the bound radioligand by increasing concentrations of the test
compounds. Data points for preliminary results were determined
in duplicate. Data points for the determination of Ki values were
measured in triplicate.
Test of Antinociceptive Activity in Mice Treated with
Formalin. Formalin injection induces a biphasic stereotypical
nocifensive behavior. Nociceptive responses are divided into an
early, short-lasting first phase (0-10 min) caused by a primary
afferent discharge produced by the stimulus, followed by a quiescent
period and then a second, prolonged phase (15-45 min) of tonic
pain. Mice (C57BL/6) were randomly assigned to one of the
experimental groups (n ) 6-8). Each mouse was placed in the
observation chamber and allowed to move freely for 15-20 min
before the start of the experiment. Mirrors were placed in order to
allow full view of the hind paws and licking of the injected paw
was recorded as nociceptive response. Four animals each received
an ip administration (6 µL/g) of PBS/DMSO (9/1) or PBS/DMSO
(99/1). Licking times between the two groups did not vary
statistically significantly (p e 0.05), and both groups were combined
as the control group. 5 (50, 10, and 5 mg/kg) dissolved in PBS/
DMSO (DMSO proportions 10, 2, and 1%), 6 (275, 55, and 27.5
mg/kg) dissolved in PBS/DMSO (DMSO proportions 10, 2, and
1%), and paracetamol (200 mg/kg) dissolved in PBS/DMSO (9/1)
were administered ip in a volume of 6 µL/g. Mice received formalin
(5%, 20 µL) in the dorsal surface of the left hind paw 15 min after