2704 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Clutterbuck et al.
d4) 158.8, 154.7, 80.7, 41.4, 28.7; LC-MS-EI 190.2 (MH+, 100);
found (CI) 190.118 79. C7H15N3O3 (MH+) requires 190.119 16.
tert-Butyl 2-(1H-Indazole-3-carbonyloxyimino)-2-aminoethyl-
carbamate (13). To a round-bottomed flask was added indazole-
3-carboxylic acid (21.1 g, 0.13 mol) in dimethylformamide (DMF)
(500 mL) under nitrogen. Carbodiimidazole (CDI) (23.2 g, 0.14
mol) was added to the yellow solution and was stirred at room
temperature for 30 min. N-(tert-Butoxycarbonyl)-2-aminoacetami-
doxime 12 (27.1 g, 0.14 mol) in DMF (100 mL) was then added,
and the solution was stirred for 18 h at room temperature. The
solvent was removed in vacuo on a high vacuum pump, and the
crude solid was dissolved in dichloromethane. The resulting
precipitate was filtered off and dried in vacuo to give a beige powder
(39.6 g, 92%): mp 185-186 °C; δH (300 MHz, MeOH-d4) 1.47
(9H, s, OtBu), 3.86 (2H, s, CH2), 7.33 (1H, t J 7.9 Hz, ArH), 7.50
(1H, t J 6.9 Hz, ArH), 7.65 (1H, d J 8.5 Hz, ArH), 8.23 (1H, d J
8.2 Hz, ArH); δC (75.5 MHz, DMSO-d6) 161.3, 157.6, 155.8, 140.8,
134.9, 126.6, 122.6, 122.1, 121.1, 110.9, 78.4, 40.9, 28.1; LC-MS-
EI (MH+, 100); found (FAB) 356.132 84. C15H19N5O4Na (M +
Na) requires 356.133 47.
3-[3-tert-Butoxycarbonylaminomethyl-1,2,4-oxadiazol-5-yl]inda-
zole (14). Boc-protected O-acylated amidoxime (13) (2.0 g, 6mmol)
was added to a 20 mL microwave vial with DMF (12 mL) and
sodium acetate (0.54 g, 6.6 mmol). The microwave reaction mixture
was heated to 140 °C for 20 min with stirring. The solvent was
then removed in vacuo on a high vacuum pump, and the remaining
crude yellow solid was dissolved in dichloromethane (100 mL) and
washed with water (100 mL), sodium hydrogen carbonate (aq) (100
mL), 1 N HCl (aq) (100 mL), and brine (100 mL). The organic
phase was then dried over magnesium sulfate and the solvent
removed in vacuo to give a white powder (1.48 g, 78%): mp
169-170 °C; δH (300 MHz, MeOH-d4) 1.47 (9H, s, OtBu), 4.55
(2H, s, CH2), 7.36 (1H, t J 6.9 Hz, ArH), 7.53 (1H, t J 5.9 Hz,
ArH), 7.68 (1H, d J 8.5 Hz, ArH), 8.26 (1H, d J 7.3 Hz, ArH); δC
(75.5 MHz, MeOH-d4) 172.6, 158.3, 142.6, 131.8, 128.7, 124.4,
122.9, 121.9, 111.9, 80.8, 37.4, 28.7; LC-MS-EI 316.2 (MH+, 100);
found (FAB) 316.140 04 C15H17N5O3 (MH+) requires 316.140 96.
Anal. Calcd for C15H17N5O3: C, 57.13; H, 5.43; N, 22.21. Found:
C, 57.51; H, 5.51; N, 21.83.
General Method for the Synthesis of the N1-Benzyl-Substituted
Oxadiazoles. To a solution of 14 (0.25 g, 0.79 mmol) in anhydrous
DMF (5 mL) was added cesium carbonate (0.78 g, 2.4 mmol),
which was stirred for 30 min. The benzyl halide was then added
(2.0 mmol), and the resulting solution was stirred overnight at room
temperature under nitrogen. The solvent was removed in vacuo and
the crude residue dissolved in ethyl acetate (20 mL). The organic
phase was washed with water (20 mL) and brine (20 mL) and dried
over maganesium sulfate. The solvent was removed in vacuo to
give the crude product. Unless otherwise stated, the crude com-
pounds were purified using column chromatography, eluting with
4:1 cyclohexane/ethyl acetate.
tert-Butyl (5-(1-Benzyl-1H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)m-
ethylcarbamate (15). White powder (0.11 g, 34%); mp 80-81 °C;
δH (300 MHz, CDCl3) 1.48 (9H, s, (OtBu), 4.61 (2H, d J 5.4 Hz,
CH2NH), 5.75 (2H, s, NCH2), 6.71-7.46 (5H, m, ArH), 8.31 (1H,
d J 8.3 Hz, ArH); δC (75.5 MHz, CDCl3) 171.4, 168.4, 162.6, 155.6,
140.5, 135.4, 130.0, 128.0, 127.6, 127.4, 123.7, 123.0, 121.6, 110.3,
80.1, 54.2, 36.5, 28.2; LC-MS-EI 406.4 (MH+, 100); found (CI)
406.187 02. C22H23N5O3 (MH+) requires 406.187 91. Anal. Calcd
for C22H23N5O3: C, 65.17; H, 5.72; N, 17.27. Found C, 65.09; H,
5.96; N, 17.06.
Unless otherwise stated, the crude compounds were purified using
column chromatography (4:1 cyclohexane/ethyl acetate).
tert-Butyl (5-(2-Benzyl-2H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)m-
ethylcarbamate (16). The compound was purified by recrystallizing
from methanol to give a white powder (0.09 g, 28%) or alternatively
column chromatography using 2:1 cyclohexane/ethyl acetate: mp
153-154 °C; δH (300 MHz, CDCl3) 1.49 (9H, s, (OtBu), 4.60 (2H,
d J 5.8 Hz, CH2NH), 6.23 (2H, s, NCH2), 7.30-7.43 (7H, m, ArH),
7.86 (1H, d J 8.4 Hz, ArH), 8.19 (1H, d J 8.2 Hz, ArH); δC (75.5
MHz, CDCl3) 168.3, 168.2, 156.3, 148.2, 135.7, 128.9, 128.6, 128.2,
127.9, 127.1, 125.8, 123.5, 120.5, 119.0, 118.5, 81.4, 56.6, 53.4,
28.3; LC-MS-EI 406.4 (MH+, 100); found (EI) 405.179 40.
C22H23N5O3 (M) requires 405.180 08.
General Method for the Deprotection of the Boc-Protected
Oxadiazoles. The boc-protected oxadiazoles (0.03 g-0.25 g) were
dissolved in trifluoroacetic acid (1.9 mL, 95%), and then triiso-
propylsilane (0.05 mL, 2.5%) and water (0.05 mL, 2.5%) were
added. The solution was allowed to stir for 18 h at room
temperature. Ice-cold diisopropyl ether or diethyl ether was added
until a white precipitate started to form. The white precipitate was
then filtered off and allowed to dry.
(5-(1-Benzyl-1H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)metha-
namine (11). White powder (60 mg, >99%): mp 118-119 °C; δH
(300 MHz, MeOH-d4) 4.39 (2H, s, CH2NH2), 5.82 (2H, s, NCH2),
(8H, m, ArH), 8.31 (1H, d J 8.3 Hz, ArH); δC (75.5 MHz, MeOH-
d4) 173.1, 166.6, 142.1, 137.3, 130.4, 129.9, 129.2, 129.0, 128.6,
125.1, 124.2, 122.2, 112.0, 54.8, 36.1; LC-MS-EI 306.4 (MH+,
100); found (CI) 306.135 01. C17H15N5O (MH+) requires 306.135 48.
Anal. Calcd for C17H15N5O: C, 66.87; H, 4.95; N, 22.94. Found:
C, 67.00; H, 4.82; N, 22.97.
(5-(2-Benzyl-2H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)metha-
namine (38). White powder (96 mg, 98%); mp 144-145 °C; δH
(300 MHz, MeOH-d4) 4.48 (2H, t J Hz, CH2NH2), 6.22 (2H, t J
Hz, NCH2), 7.29-7.47 (7H, m, ArH), 7.80 (1H, m, ArH), 8.18
(1H, m, ArH); δC (75.5 MHz, MeOH-d4) 170.3, 166.4, 149.5, 137.4,
129.8, 129.2, 128.6, 127.3, 124.8, 121.4, 120.0, 119.4, 57.6, 36.0;
LC-MS-EI 306.4 (MH+, 75); found (CI) 306.135 54. C17H15N5O
(MH+) requires 306.135 48.
Biological Methods. Preparation of Rat Forebrain Synapto-
somes and Homogenates. Experiments were performed using
forebrain (whole brain less cerebellum/medulla) from male Wistar
rats weighing 175-250 g. All efforts were made to reduce the
number of animals used, and all experiments were carried out in
accordance with the U.K. Animals (Scientific Procedures) Act,
1986, and the European Community Council Directive of November
24, 1986 (86/609/EEC). Following killing of animals by stunning
and decapitation, the forebrain was rapidly dissected and transferred
to a weighed tube containing ice-cold 0.25 M sucrose.
Synaptosomes (heavy and light mitochondrial fraction containing
synaptosomes) were prepared by transferring the forebrain (of
known wet weight) to a glass Potter vessel to which 9 volumes of
ice-cold 0.25 M sucrose had been added and homogenizing, using
a Teflon pestle, by 8 “up and down strokes” of a Braun Potter S
motor driven homogenizer set to 900 rpm. The resulting homoge-
nate was centrifuged at 1036g at 4 °C for 10 min and the supernatant
collected. The remaining pellet was resuspended, as above, in fresh
ice-cold 0.25 M sucrose and the centrifugation step repeated. The
supernatant fractions were pooled and centrifuged at 40000g
(average) at 4 °C for 15 min and the resulting pellet resuspended
in the appropriate assay buffer at a concentration of 20-25 mg
wet weight per mL of appropriate assay buffer.
General Method for the Mitsunobu Synthesis of the N2-Benzyl-
Substituted Oxadiazoles. To a solution of 14 (0.25 g, 0.79 mmol),
under nitrogen, in toluene (10 mL), benzyl alcohol (0.87 mmol),
tributylphosphine (0.26 g, 0.31 mL, 1.26 mmol), and TMAD (0.22
g, 1.26 mmol) were added successively. The resulting clear-yellow
solution was allowed to stir overnight at room temperature. The
solution was than extracted with water, 1 N HCl, 1 N sodium
hydroxide (aq), and brine. The combined aqueous phases were
washed with dichloromethane and the combined organic phases
dried over magnesium sulfate. The solvent was removed in vacuo.
Homogenates were prepared by transferring the known weight
of forebrain to a cooled tube containing 9 volumes of ice-cold 50
mM HEPES buffer, pH 7.4. The mixture was homogenized at 4
°C using 3 × 5 s bursts of an Ultra-Turrax homogenizer set at
maximum speed. The resulting homogenate was centrifuged at
40000g (average) at 4 °C for 15 min and the supernatant discarded.
The resulting pellet was resuspended in 9 volumes of fresh ice-
cold pH 7.4 buffer (as above), the centrifugation step was repeated
and the resulting pellet resuspended in the appropriate assay buffer
at a concentration of 20-25 mg wet weight per mL.