T. M. Shoup et al. / Tetrahedron Letters 55 (2014) 682–685
683
A
B
O
.HCl
NH
a,b,c
d
HO
HO
*
HO
*
N
N
O
O
O
[1-11C]-(+)-PHNO
(+)-HNO
O
O
*
*
e
f
TIPSO
HO
TIPSO
N
N
N
O
O
O
[3-11C]-(+)-PHNO
1
Scheme 1. (A) Radiosynthesis of [1-11C]-(+)-PHNO3: (a) EtMgBr, [11C]CO2, (b) phthaloyl dichloride/2,6-di-tert-butylpyridine, (c) distill; THF, N,N-diisopropylethylamine,
60 °C, (d) 1 M LiAlH4, then HCl; (B) [3-11C]-(+)-PHNO10: (e) lithium hexamethyldisilazide (LHMDS), [11C]CH3I, THF, 0 °C; (f) 1 M LiAlH4, 60 °C, then AcOH, 60 °C. Asterisk
denotes site of 11C.
dopamine receptors in brown adipose tissue (BAT; aka brown fat)
in rat.
radiochemical yield)10 for consistent preparation starting from 1.
Carbon-11 labeling was achieved by 11C-methylation of the corre-
sponding enolate of 1 generated by hindered base lithium hexam-
ethyldisilazide (LHMDS; 1 M in THF).10 We have found that
commercial bottles of this reagent contain unsuitable amounts of
base impurity, most likely formed from LHMDS induced decompo-
sition of THF that reacts with [11C]CH3I to give a polar 11C-labeled
side-product in 40–99% radiochemical yield. In contrast, use of
commercial solutions of 1 M LHMDS in hexanes, which are clear
and colorless, consistently reduces variability by decreasing side-
product formation to 15–40%.
Synthesis of labeling precursor 1 involves 12 steps (see ESI for
details) and uses modifications of the method originally described
by Jones et al.1 for the preparation of PHNO and subsequent proce-
dures of Perone et al.12 and Delgado et al.13 Starting with 7-meth-
oxy-1-tetralone, demethylation using aluminum trichloride in
toluene gave 7-hydroxy-1-tetralone (2) in 90% yield which was
converted to 7-benzyloxy-1-tetralone (3) using benzyl bromide
in 83% yield. It was worthy of note that the benzyloxy derivative
provided superior yield in the subsequent deprotection reactions
on compound 10 compared with alternative protecting groups,
including methyl ether. Compound 3 was treated with hydroxyl-
amine hydrochloride in dry pyridine which afforded the desired
hyroxyiminotetralone (4) in high yield. Neber rearrangement12
was used to synthesize 2-amino-1-tetralone 6 starting from the
tosyloxy derivative 5. Stereoselective reduction of 6 via sodium
borohydride gave the corresponding trans-2-amino-1-tetraol 7 as
the major product.14 The trans-amino tetralone 7 was acylated
with chloroacetyl chloride and cyclized with NaH in THF. Reduc-
tion of the benzoxazine-3-one 9 with LiAlH4 afforded the desired
trans-benzoxyoxazine 10. Debenzylation by catalytic hydrogena-
tion gave racemic HNO (see Scheme 2).
Resolution of HNO enantiomers was performed on an AD-H col-
umn by supercritical fluid chromatography (SFC). Products were
analyzed by UltraPerformance Convergence Chromatography
(UPC2, Waters; see ESI for details). An extensive screening of chiral
stationary phases indicated that the CHIRALPAK AD-H column of-
fered a desirable separation of the HNO racemate in less than
6 min, as shown in Figure 1A. Compared to the separation by HPLC,
the SFC method presented here was three times faster, resulting in
a three-fold productivity improvement when scaled up to the pre-
parative scale. The productivity was further improved by employ-
ing stacked injections; performed under isocratic conditions,
injections are made during the course of chromatography such that
the first peak from a subsequent injection elutes off the column
adjacent to the last peak of the preceding injection, without com-
promising chromatographic efficiency. A total of 1 g of the race-
mate was purified in less than 2 h. Post-purification analysis
showed that the (+)-isomer had an enantiomeric excess (ee)
>99% (Fig. 1B). A white solid of (+)-HNO was obtained after drying
followed by trituration with hexanes.
Using our modified manual radiosynthesis procedure for
[3-11C]-(+)PHNO, [11C]CH3I (prepared via [11C]CO2 using a GE
Tracerlab FXM module) was trapped in a vial cooled to 0 °C contain-
ing 1 (3 mg) in THF (200
lL). Subsequently, a solution of 1 M
LHMDS in hexanes (20 L) was then added to the vial. The reaction
l
mixture was held at 0 °C for 3 min and then allowed to warm to
25 °C over 3 min, at which time the mixture was quenched using
0.2 M anhydrous methanol in THF (80 lL). Analytical HPLC analy-
sis10 of aliquots from reaction mixtures showed conversion to
the 11C-labeled amide was between 60% and 85%. Amide reduction
was achieved with 1 M LiAlH4 in THF (100 lL) followed by heating
at 60 °C for 7 min. The mixture was quenched at 60 °C with 0.5 mL
of 50% acetic acid for dissolving salts and removing the silyl pro-
tecting group concurrently. [3-11C]-(+)PHNO was isolated by re-
verse-phase HPLC (Phenomenex Luna 10
l
C18, 250 Â 10 mm,
20% acetonitrile 80% 0.1 M ammonium formate (pH 4.5 adjusted
with acetic acid). The desired fraction (6 mL/min, tR = 7 min) was
collected and diluted with 30 mL of water containing 60 mg of so-
dium carbonate. This solution was passed through a solid phase
extraction cartridge (SPE; C18 SepPakÒ; Waters) which was fol-
lowed by washing with 10 mL of water. [11C]-(+)PHNO was eluted
from the SPE with 1 mL of ethanol into a vial containing 10 mL of
0.9% saline and the resulting solution was filtered through a
0.22 l
m sterile syringe filter (MillexÒGV; Millipore). Synthesis
was complete within 40 min with more stable radiochemical
yields (10–20% uncorrected relative to [11C]CH3I, n = 3) and consis-
tent specific activities suitable for clinical use (38–84 GBq/lmol;
1.0–2.3 Ci/mol at end of synthesis) were achieved with high
radiochemical purities (>99%). In future, an automated radiosyn-
thesis with this revised procedure would enable higher starting
activities of [11C]CO2 to be used and consequently higher specific
activities to be achieved. This will ensure that pharmacological ef-
fects caused by this agonist radiotracer are avoided.
The (+)-HNO isomer was O-silylated using 2.5 equiv of triiso-
propylsilyl chloride (TIPSCl) and imidazole in THF to yield 11. Acet-
ylation with acetic anhydride and triethylamine gave the protected
acetyl amide 1. (+)-PHNO was also prepared from (+)-HNO using
iodopropane in 73% yield and >99% ee.
A preliminary PET imaging study with [3-11C]-(+)-PHNO was
conducted in rat in order to determine whether this tracer is suit-
able for imaging BAT, (brown fat). Dopamine receptors are known
to be expressed in BAT.15,16 The thermogenic activity of BAT is
modulated by dopaminergic compounds15,17 and may play a role
The one-pot radiosynthesis for [3-11C]-(+)PHNO was optimized
from the variable literature procedure (2–30% uncorrected