4598 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Pinkerton et al.
hydroxy-2-propylphenoxy)butoxy]benzonitrile as a white solid.
4-[4-(4-Acetyl-3-hydroxy-2-propylphenoxy)butoxy]benzoni-
trile (5.76 g, 15.7 mmol), trimethylsilyl azide (3.61 g, 4.16 mL,
31.3 mmol) and dibutyltin oxide (586 mg, 2.4 mmol) were
dissolved in toluene (130 mL) and heated to reflux for 16 h.
The reaction mixture was then cooled to room temperature
and applied directly to a silica gel column (eluting first with
20% ethyl acetate/hexanes followed by 10% MeOH/dichlo-
romethane) to give 6.09 g of 1-(2-hydroxy-3-propyl-4-{4-[4-(2H-
tetrazol-5-yl)phenoxy]butoxy}phenyl)ethanone (95%) as a white
solid. Compound was >95% pure by HPLC and NMR analysis.
1H NMR (DMSO-d6, 500 MHz), δ 12.85 (s, 1H, OH), 7.95 (d,
2H), 7.80 (d, 1H), 7.11 (d, 2H), 6.66 (d, 1H), 4.18-4.14 (m, 4H),
2.58-2.51 (m, 5H), 1.95-1.90 (m, 4H), 1.49-1.44 (m, 2H), 0.86
(t, 3H). MS (ESI): 411.2 (M + H)+,Calcd 411.2. HRMS calcd
for C22H27N4O4 (M + H)+: 411.2032. Found: 411.2000. Anal.
(C22H26N4O4‚H2O) C, H, N.
1-(2-Hyd r oxy-3-m eth yl-4-{4-[4-(2H-tetr a zol-5-yl)-p h en -
oxy]-b u t oxy}-p h en yl)-3-m et h yl-b u t a n -1-on e (9). Isobu-
tyryl chloride (1.25 g, 1.3 mL, 10.4 mmol) was added to a
stirred solution of 2-methylresorcinol (1 g, 8.0 mmol) and
aluminum trichloride (1.39 g, 10.4 mmol) in dichloromethane
(40 mL) at 0 °C. The reaction was allowed to warm to room
temperature, then stirred for 16 h. It was then quenched by
addition of 1 N aqueous HCl. The organic layer was separated,
dried over MgSO4 and then concentrated in vacuo to give a
residue that was purified via column chromatography on silica
gel (eluting 5-60% ethyl acetate/hexanes) to give 946 mg (57%)
of 1-(2,4-dihydroxy-3-methyl-phenyl)-3-methyl-butan-1-one as
a white solid. Potassium carbonate (398 mg, 2.88 mmol) was
added to a stirred solution of 1-(2,4-dihydroxy-3-methyl-
phenyl)-3-methyl-butan-1-one (300 mg, 1.44 mmol) and 4-(4-
bromo-butoxy)-benzonitrile (403 mg, 1.58 mmol) in acetone (20
mL) at 45 °C. The reaction mixture was stirred for 16 h, then
the acetone was removed in vacuo. The residue was then mixed
with dichloromethane (100 mL) and water (100 mL). The
organic layer was separated, dried over MgSO4 and then
concentrated in vacuo to give a residue that was purified via
column chromatography on silica gel (eluting 5-50% ethyl
acetate/hexanes) to give 378 mg (69%) of 4-{4-[3-hydroxy-2-
methyl-4-(3-methyl-butyryl)-phenoxy]-butoxy}-benzonitrile as
a white solid. 4-{4-[3-Hydroxy-2-methyl-4-(3-methyl-butyryl)-
phenoxy]-butoxy}-benzonitrile (257 mg, 0.67 mmol), trimeth-
ylsilyl azide (155 mg, 0.18 mL, 1.3 mmol) and dibutyltin oxide
(25 mg, 0.10 mmol) were dissolved in toluene (12 mL) and
heated to reflux for 16 h. The reaction mixture was then cooled
to room temperature and applied directly to a silica gel column
(eluting first with 20% ethyl acetate/hexanes followed by 10%
MeOH/dichloromethane) to give 242 mg of 1-(2-hydroxy-3-
methyl-4-{4-[4-(2H-tetrazol-5-yl)-phenoxy]-butoxy}-phenyl)-3-
methyl-butan-1-one (85%) as a white solid. Compound was
>95% pure by HPLC and NMR analysis. 1H NMR (DMSO-d6,
500 MHz), δ 13.03 (s, 1H), 7.95 (d, 2H), 7.85 (d, 1H), 7.10 (d,
2H), 6.65 (d, 1H), 4.18-4.13 (m, 4H), 2.85 (d, 2H), 2.17-2.14
(m, 1H), 2.00 (s, 3H), 1.95-1.93 (m, 4H), 0.93 (d, 6H). MS
(ESI): 425.2 (M + H)+, calcd 425.2. HRMS calcd for C23H29N4O4
(M + H)+: 425.2189. Found: 425.2162. Anal. (C23H28N4O4‚
H2O) C, H, N.
1-(2-Hyd r oxy-3-m eth yl-4-{4-[4-(2H-tetr a zol-5-yl)-p h en -
oxy]-bu toxy}-p h en yl)-p r op a n -1-on e (7). Prepared in
a
similar fashion as outlined for compound 9 using propanoic
1
acid chloride. H NMR (DMSO-d6, 500 MHz) δ 12.70 (s, 1H),
7.95 (d, 2H), 7.83 (d, 1H), 7.13 (d, 2H), 6.66 (d, 1H), 4.18-4.13
(m, 4H), 2.04 (q, 2H), 1.99 (s, 3H), 1.97-1.92 (m, 4H), 1.10 (t,
3H). MS (ESI) 396.7 (M+ + H), Calcd 396.5. Anal. (C21H24N4O4‚
H2O) C, H, N.
1-(2-Hyd r oxy-3-m eth yl-4-{4-[4-(2H-tetr a zol-5-yl)-p h en -
oxy]-bu toxy}-p h en yl)-bu ta n -1-on e (8). Prepared in a simi-
lar fashion as outlined for compound 9 using butyryl chloride
1H NMR (DMSO-d6, 500 MHz), δ 13.02 (s, 1H), 7.96 (d, 2H),
7.85 (d, 1H), 7.14 (d, 2H), 6.66 (d, 1H), 4.18-4.15 (m, 4H), 2.98
(t, 2H), 2.00 (s, 3H), 1.96-1.93 (m, 4H), 1.67-1.63 (m, 2H),
0.95 (t, 3H). MS (ESI): 411 (M + H)+, Calcd 411.2. Anal.
(C22H26N4O4‚H2O) C, H, N.
1-(2-Hyd r oxy-3-p r op yl-4-{4-[4-(2H-tetr a zol-5-yl)-p h en -
oxy]-b u t oxy}-p h en yl)-3-m et h yl-b u t a n -1-on e (10). Pre-
pared in a similar fashion as outlined for compound 4 using
1-(2,4-Dihydroxy-3-propyl-phenyl)-3-methyl-butan-1-one. 1H
NMR (DMSO-d6, 500 MHz) δ 13.03 (s, 1H), 7.97 (d, 2H), 7.85
(d, 1H), 7.15 (d, 2H), 6.65 (d, 1H), 4.17-4.14 (m, 4H), 2.85 (d,
2H), 2.51 (t, 2H), 2.17-2.12 (m, 1H), 1.94-1.92 (m, 4H), 1.48-
1.43 (m, 2H), 0.94 (d, 6H), 0.86 (t, 3H). MS (ESI) 452.5 (M+
1), Calcd 452.6. Anal. (C25H32N4O4‚H2O) C, H, N.
+
In Vitr o Bin d in g Stu d ies. Ma ter ia ls. Glutamate, GDP,
probenecid and GTPγS were obtained from Sigma Chemical
(St Louis, MO). [35S]GTPγS and 14 were obtained from Tocris
(Ellisville, MO).
Mem br a n e P r ep a r a tion . hmGlu2 and hmGlu3 receptor-
expressing stable cell lines were grown to confluence in a
T-225-cm2 flask and washed twice with ice-cold PBS. The cells
were then scraped with a cell scraper in phosphate-buffered
saline and harvested by centrifugation (200g) using a tabletop
centrifuge. The cell pellet was homogenized in hypotonic buffer
A (20 mM HEPES and 10 mM EDTA, pH 7.4) using a Polytron
homogenizer (Brinkmann, Westbury, NY). The homogenate
was centrifuged at 40 000g for 20 min. The resulting pellet
was washed once in the same buffer and once with buffer B
(20 mM HEPES and 0.1 mM EDTA, pH 7.4). At the last
centrifugation, the pellet was resuspended in buffer B and the
homogenate was aliquoted and stored at -80 °C at a protein
concentration of approximately 1 mg/mL. Protein measure-
ment was determined with the Bio-Rad detergent-compatible
protein assay kit using bovine serum albumin as standard.
Rats (250-300 g) were decapitated; the whole brain was
removed, placed on ice, and homogenized in 6 volumes (w/v)
of 10% sucrose at 4 °C using a glass-Teflon homogenizer. The
homogenate was centrifuged at 1000g for 10 min, and the
supernatant was centrifuged at 40 000g for 20 min at 4 °C.
The supernatant was removed and the pellet was resuspended
in buffer C (5 mM HEPES-KOH, pH 7.4). The homogenate
was freeze-thawed twice before being centrifuged at 40 000g
for 20 min. Finally, the resulting pellet was resuspended in
buffer C, aliquoted and stored at -70 °C until used.
[
35S]GTP γS Bin d in g Assa y. Membranes were thawed and
homogenized in 25 mL of a 20 mM HEPES containing 0.1 mM
EDTA, pH 7.4, and centrifuged at 40 000g for 20 min. The
pellet was resuspended in assay buffer containing 20 mM
HEPES, pH 7.4, 100 mM NaCl and 3 mM MgCl2 at a final
protein concentration of 0.5 mg/mL (hmGlu2 and hmGlu3
1-(2-H y d r o x y -3-m e t h y l-4-{4-[4-(2H -t e t r a zo l-5-y l)-
p h en oxy]bu toxy}p h en yl)eth a n on e (5). Prepared in a simi-
lar fashion as outlined for compound 4 using 1-(2,4-dihydroxy-
3-methylphenyl)ethanone. 1H NMR (DMSO-d6, 500 MHz), δ
12.85 (s, 1H, -OH), 7.95 (d, 2H), 7.80 (d, 1H), 7.11 (d, 2H),
6.66 (d, 1H), 4.18-4.14 (m, 4H), 2.57 (s, 3H), 2.01 (s, 3H), 1.95-
1.90 (m, 4H). MS (ESI): 382.4 (M + H)+, Calcd 382.4. Anal.
(C20H22N4O4‚H2O) C, H, N.
1-(2-Hydr oxy-3-pen tyl-4-{4-[4-(2H-tetr azol-5-yl)ph en oxy]-
bu toxy}p h en yl)eth a n on e(6). Prepared in a similar fashion
as outlined for compound 4 using 1-(2,4-dihydroxy-3-pen-
tylphenyl)ethanone. 1H NMR (DMSO-d6, 500 MHz), δ 12.84
(s, 1H, -OH), 7.98 (d, 2H), 7.82 (d, 1H), 7.13 (d, 2H), 6.65 (d,
1H), 4.17-4.14 (m, 4H), 2.58-2.52 (m, 5H), 1.97-1.91 (m, 4H),
1.55-1.34 (m, 6H), 0.87 (t, 3H). MS (ESI): 438.6 (M+H)+,
Calcd 438.5. Anal. (C24H30N4O4‚H2O) C, H, N.
receptors) or 0.1 mg/mL (rat brain). In
a 96-well plate
(Beckman Coulter, Fullerton, CA), test compounds and
glutamate were added along with 5 µM GDP, membrane (10
µg/well for rat brain and 50 µg for recombinant mGlu recep-
tors), and 0.05 nM [35S]GTPγS to achieve a total volume of
0.5 mL in assay buffer. The plate was incubated at 30 °C for
1 h, then the assay was terminated by rapid filtration over
Unifilter GF/B plate using a 96-well cell harvester (Brandel,
Gaithersburg, MD). The plate was rinsed three times with ice-
cold assay buffer, dried, and 50 µL of Microscint 20 was added
to each well. The plate was counted in a Topcount scintillation
counter (PerkinElmer Life Science). Each experiment was
performed using triplicate samples per data point and then