4518 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Notes
The organic layer was dried (MgSO4) and concentrated by
rotary evaporation, and the residue was chromatographed on
silica gel using 2:1 diethyl ether-petroleum ether as eluent
to afford 108 mg (85%) of the title compound 7: Rf ) 0.11 (10%
MeOH-CHCl3); 1H NMR δ (CDCl3) 5.36 (m, 8H), 3.90 (t, J )
8.38 Hz, 2H), 3.78 (t, J ) 8.38 Hz, 2H), 3.55 (t, J ) 8.38 Hz,
2H), 3.50 (t, J ) 8.38 Hz, 2H), 2.80 (m, 6H), 2.41 (t, J ) 8.20
Hz, 2H), 2.10 (m, 4H), 1.76 (m, 2H), 1.32 (m, 6H), 0.90 (t, J )
6.50 Hz, 3H). Anal. (C24H41NO3) C, H, N.
N-[2-H yd r oxy-1-(h yd r oxym et h yl)et h yl]a r a ch id on yl-
a m id e (8). Arachidonic acid chloride, prepared from 100 mg
(0.33 mmol) of arachidonic acid as described under compound
5, was dissolved in 3 mL of anhydrous methylene chloride.
and a solution of 60 mg (0.6 mmol) of 2-amino-1,3-propanediol
(serinol) in 2 mL of dry pyridine was added portionwise. After
the mixture was stirred at room temperature for a further 15
min, the yellow precipitate formed was filtered off and washed
with methylene chloride (10 mL). The combined filtrate and
washings were concentrated in vacuo, and the residue was
dissolved in 10 mL of methylene chloride and washed succes-
sively with 10% HCl, 10% NaOH, and water. The organic
layer was dried (MgSO4), and the solvents were removed by
rotary evaporation. The residue was chromatographed on
silica gel using 2% MeOH-CHCl3 as eluent to afford 60 mg
(48%) of the title compound 8: 1H NMR (CDCl3) δ 6.19 (br s,
1H), 5.30 (m, 8H), 3.91-3.99 (m, 1H), 3.82 (d, J ) 4.6 Hz, 4H),
2.79-2.84 (m, 6H), 2.15-2.29 (t, J ) 7.5 Hz, 2H), 2.00-2.14
(m, 4H), 1.70-1.81 (m, 2H), 1.25-1.39 (m, 6H), 0.89 (t, J )
6.5 Hz, 3H). Anal. (C23H39NO3) C, H, N.
4H), 5.35 (m, 8H), 2.80 (m, 6H), 2.43 (t, J ) 7.76 Hz, 2H), 2.21-
1.99 (m, 4H), 1.80 (m, 2H), 1.34 (m, 6H), 0.88 (t, J ) 6.73 Hz,
3H). Anal. (C26H37NO2) C, H, N.
N-(3-Hyd r oxyp h en yl)a r a ch id on yla m id e (12). The title
compound was synthesized from 100 mg (0.33 mmol) of
arachidonic acid and 3-aminophenol as described under 11 to
afford 86 mg (61%) of compound 12: 1H NMR (2:1 DMSO-
CDCl3) δ 8.70 (s, 1H), 8.04 (s, 1H), 7.32 (s, 1H), 7.14 (m, 1H),
6.67 (m, 1H), 6.46 (m, 1H), 5.36 (m, 8H), 2.80 (m, 6H), 2.39 (t,
J ) 7.71 Hz, 2H), 2.10 (m, 4H), 1.83 (m, 2H), 1.29 (m, 6H),
0.88 (t, J ) 6.75 Hz, 3H). Anal. (C26H37NO2) C, H, N.
N-(4-Hyd r oxyp h en yl)a r a ch id on yla m id e (13). The title
compound was synthesized from 100 mg (0.33 mmol) of
arachidonic acid and 4-aminophenol as described under 11 to
afford 96 mg (68%) of compound 13: 1H NMR (2:1 DMSO-
CDCl3) δ 9.51 (s, 1H), 9.00 (s, 1H), 7.34 (d, 2H), 6.65 (d, 2H),
5.33 (m, 8H), 2.80 (m, 6H), 2.26 (t, J ) 7.50 Hz, 2H), 2.06 (m,
4H), 1.66 (m, 2H), 1.28 (m, 6H), 0.87 (t, J ) 6.76 Hz, 3H). Anal.
(C26H37NO2) C, H, N.
N-(4-Hyd r oxyben zyl)a r a ch id on yla m id e (14). A solu-
tion of arachidonic acid chloride in dry benzene, prepared from
100 mg (0.33 mmol) of arachidonic acid according to the
procedure described under 5, was cooled in an ice bath, and a
solution of 340 mg (1.65 mmol) of the tetrahydropyranyl ether
of 4-hydroxybenzylamine in 2.0 mL of anhydrous THF was
added dropwise. The reaction mixture was stirred at room
temperature for a further 15 min; then 10 mL of saturated
NaHCO3 solution and 20 mL of diethyl ether were added, the
organic layer was separated, and solvents were removed by
rotary evaporation. The residual oil was dissolved in 5 mL of
methanol, and the pH of the solution was adjusted to ap-
proximately pH 3 by dropwise addition of concentrated hy-
drochloric acid. The solution was warmed in a water bath to
50 °C for 10 min and then cooled to room temperature,
saturated NaHCO3 solution was added to raise the pH to
approximately pH 8, and most of the solvents were removed
by rotary evaporation. The residue was dissolved in diethyl
ether, and the solution was dried (MgSO4). The ether was
removed by rotary evaporation. The crude product was
purified by column chromatography on silica gel (80:20 diethyl
ether-petroleum ether) to afford 110 mg (82%) of the title
amide 14: Rf ) 0.29 (80:20 diethyl ether-petroleum ether);
1H NMR δ (CDCl3) 7.08 (d, J ) 8.50 Hz, 2H), 6.79 (d, J ) 8.50
Hz, 2H), 5.95 (t, J ) 5.33 Hz, 1H), 5.36 (m, 8H), 4.33 (d, J )
5.56 Hz, 2H), 2.80 (m, 6H), 2.28-1.98 (m, 6H), 1.73 (m, 2H),
1.28 (m, 6H), 0.88 (t, J ) 6.78 Hz, 3H). Anal. (C26H37NO2) C,
H, N.
Ra d ioliga n d Bin d in g Assa y. For CB1, rat forebrain
membranes were prepared according to the procedure of
Devane et al.24 The binding of the novel probes to the
cannabinoid receptor was assessed as previously described,24,25
except that the membranes were treated with PMSF. Mem-
branes, previously frozen at -80 °C, were thawed on ice. To
the stirred suspension were added five volumes of 25 mM Tris-
HCl Buffer, pH 7.4, 5 mM MgCl2, and 1 mM EDTA (TME)
containing 50 µM PMSF (made fresh daily in 2-propanol as a
10 mM stock). After 30 min the membranes were pelleted,
the supernatant was discarded, and the pellet was resus-
pended in five volumes of the PMSF containing buffer. At the
end of the second 30 min incubation, the membranes were
pelleted and washed three times with TME to remove unre-
acted PMSF. The treated membranes were subsequently used
in the binding assay described below. Approximately 50 µg
of PMSF-treated membranes were incubated in silanized
culture tubes with TME containing 0.1% essentially fatty acid
free bovine serum albumin (BSA), 0.8 nM [3H]CP-55,940, and
various concentrations of the anandamide analogs in a final
volume of 200 µL. Assays were incubated for 1 h at 30 °C
and terminated by the addition of 250 µL of TME containing
5% BSA. The tubes were immediately filtered using GF/C
filters on a Brandell M-24 cell harvester. Following four
washes with ice-cold wash buffer (20 mM Tris-HCl, pH 7.4, 3
mM MgCl2, 4 mL/well), the filters were collected, shaken for
1 h with 2 mL of 0.1% sodium dodecyl sulfate, and counted in
a liquid scintillation counter to determine the amount of bound
radioligand. Nonspecific binding was assessed using 250 nM
N-(2-Hyd r oxy-2-eth yleth yl)a r a ch id on yla m id e (9).
A
solution of arachidonic acid chloride was prepared from 100
mg (0.33 mmol) of arachidonic acid as described under 5, and
then a solution of (()-1-amino-2-butanol (293 mg, 3.3 mmol)
in 2 mL of dry methylene chloride was added. After a further
15 min of stirring at room temperature, the reaction mixture
was diluted with 10 mL of methylene chloride, washed
successively with 10% HCl and 10% NaOH, and dried (MgSO4).
The solvent was removed by rotary evaporation, and the
residue was purified by column chromatography using 30%
petroleum ether-diethyl ether as eluent to yield 65 mg (52%)
of the title compound 9: 1H NMR (CDCl3) δ 0.88 (t, J ) 6.50
Hz, 3H), 0.96 (t, J ) 7.00 Hz, 3H), 1.31 (s, 6H), 1.47 (m, 2H),
1.67-1.80 (m, 2H), 2.03-2.25 (m, 6H), 2.81 (m, 6H), 3.08-
3.17 (m, 1H), 3.43-3.62 (m, 2H), 5.30 (m, 8H), 5.90 (br s, 1H).
Anal. (C24H41NO2) C, H, N.
N-(tr a n s-2-Hyd r oxycycloh exyl)a r a ch id on yla m id e (10).
A solution of arachidonic acid chloride was prepared from 100
mg (0.33 mmol) of arachidonic acid as described under 5, and
then a solution of 2-aminocyclohexanol hydrochloride (100 mg,
0.6 mmol) in 2 mL of dry pyridine was added portionwise. After
being stirred at room temperature for a further 15 min, the
mixture was evaporated in vacuo at room temperature, and
the residue was dissolved in 10 mL of methylene chloride. The
solution was washed successively with 10% HCl, 10% NaOH,
and water. The organic layer was dried (MgSO4) and concen-
trated by rotary evaporation, and the residue was chromato-
graphed on silica gel using 2:1 diethyl ether-petroleum ether
as eluent to afford 90 mg (68%) of the title compound 10: 1H
NMR (CDCl3) δ 5.30-5.40 (m, 9H), 3.60-3.72 (m, 2H), 3.37-
3.34 (m, 1H), 2.79-2.84 (m, 6H), 1.97-2.23 (m, 8H), 1.70-
1.81 (m, 4H), 1.25-1.35 (m, 10H), 0.89 (t, J ) 6.50 Hz, 3H).
Anal. (C24H41NO2) C, H, N.
N-(2-Hyd r oxyp h en yl)a r a ch id on yla m id e (11). A solu-
tion of arachidonic acid chloride in dry benzene, prepared from
100 mg (0.33 mmol) of arachidonic acid according to the
procedure described under 5, was cooled in an ice bath, and a
solution of 360 mg (3.3 mmol) of 2-aminophenol in 4.5 mL of
anhydrous THF was added dropwise. The reaction mixture
was stirred at room temperature for a further 15 min and then
diluted with 15 mL of methylene chloride and 5 mL of water.
The organic layer was separated and dried (MgSO4), and the
solvents were removed by rotary evaporation. The residue was
purified by column chromatography on silica gel (1:1 diethyl
ether-petroleum ether) to afford 120 mg (85%) of the title
compound 11: Rf ) 0.39 (1:1 diethyl ether-petroleum ether);
1H NMR δ (CDCl3) 8.95 (s, 1H), 8.00 (s, 1H), 7.21-6.79 (m,