International Journal of Organic Chemistry, 2012, 2, 202-223
http://dx.doi.org/10.4236/ijoc.2012.23030 Published Online September 2012 (http://www.SciRP.org/journal/ijoc)
Reductive Amination with [11C]Formaldehyde: A Versatile
Approach to Radiomethylation of Amines
Chunying Wu1, Ruoshi Li1, Dorr Dearborn2, Yanming Wang1*
1Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Cleveland, USA
2Environmental Health, Case Western Reserve University, Cleveland, USA
Email: *yanming.wang@case.edu
Received February 16, 2012; revised March 24, 2012; accepted April 2, 2012
ABSTRACT
Carbon-11 radiolabeled amines constitute a very important class of radioligands that are widely used for positron emis-
sion tomography (PET) imaging. Radiolabeling of amines is often achieved through radiomethylation using [11C]CH3I
or [11C]CH3OTf under basic conditions in a strictly anhydrous environment. Functional groups such as hydroxyl and
carboxyl groups that are often present in the molecules are normally base sensitive and require protection and deprotec-
tion, which substantially prolongs and complicates the radiolabeling process. Here we report a versatile approach to a
series of C-11 radiolabeled amines prepared through reductive amination using [11C]formaldehyde. Using a variety of
substrates bearing different functional groups, we demonstrate the general utility of this method. In contrast to conven-
tional radiomethylation methods, the reductive amination using [11C]formaldehyde can be carried out in an aqueous
environment relatively quickly without the need of protection of base-sensitive functional groups.
Keywords: C-11 Formaldehyde; Radiomethylation; Reductive Amination; Positron Emission Tomography;
Radiolabelling
1. Introduction
Molecular imaging has become an indispensible tool in
biomedical research. To date, a variety of molecular im-
aging modalities have been established that complement
each other in terms of sensitivity and resolution. Among
them, positron emission tomography (PET) and magnetic
resonance imaging (MRI) are widely used for clinical
studies while optical imaging including multiphoton mi-
croscopy, bioluminescence imaging, and fluorescent
molecular tomography (FMT) are widely used for pre-
clinical studies. While each imaging modality is designed
to detect different signals ranging from high-energy
gamma rays to low-energy radiofrequency, use of these
imaging modalities largely depends on the availability of
endogenous or exogenous molecular probes carrying the
required specific signals that can be readily detected at
the targets of interest.
Thus, significant efforts have been made to develop
molecular probes in line with advancements in mechani-
cal design and optimization of imaging devices. While
different sets of chemical and pharmacological properties
are required for different modalities, development of
molecular probes for PET represents a unique challenge
in many aspects. This is largely due to the fact that PET
probes must be labeled with positron-emitting radionu-
clides such as carbon-11 or fluorine-18. The inherent
short half-lives of these positron emitters (20 min for
C-11 and 110 min for F-18) make radiolabeling a very
timeconstrained process. In addition, these radionuclides
need to be produced by a cyclotron not only in trace
quantities but also in very limited forms. For example,
C-11 labelling is normally achieved through radiomethy-
lation using [11C]methyliodide [1] or [11C]methyltriflate
[2]. As a result, the chemical reactions with [11C]CH3I or
[11C]CH3OTf must be carried out under very strict, often
air- and moisture-free conditions. Often times, a trace
amount of water present either in the solvents or reagents
reduces radiochemical yields significantly or even abol-
ishes the reactions. Other reactive functional groups such
as hydroxyl or carboxyl groups present in the precursors
also need to be protected before radiolabeling and sub-
sequently deprotected after radiolabeling, which further
prolongs radiosynthesis time and complicates the purifi-
cation process.
Radiomethylation has been the primary reaction used
to radiolabel alkylated amines, which constitute a very
important class of PET radiotracers. Various amines have
been identified as radiotracers for PET imaging of vari-
ous molecular targets [3-18]. Given the fact that most
amine radiotracers possess functional groups that are
*Corresponding author.
C
opyright © 2012 SciRes. IJOC
C. Y. WU ET AL. 203
either base-sensitive or can be readily methylated, C-11
radiolabeling of those functionalized amines must un-
dergo extensive protection and deprotection processes
before they are subjected to radiomethylation with
[11C]CH3I or [11C]CH3OTf under basic conditions. In
order to skip the protection/deprotection steps, efforts
have been made to radiolabel certain functionalized
amines based on different reactivity toward C-11 methy-
lating reagents. For example, for the radiolabeling of PIB,
direct radiomethylation was achieved in an HPLC loop
filled with unprotected precursor using [11C]CH3OTf [19].
This is because [11C]CH3OTf reacts with amino groups
faster than hydroxyl groups. However, this method is
applicable only to certain functionalized amines and is
limited to small-production.
In addition to direct methylation, another important
approach to synthesizing alkylated amines is reductive
amination, which has long been used in organic synthesis.
In this reaction, amines are first carbonylated with alde-
hydes, ketones, or carboxylates to form imines followed
by subsequent reduction. To apply this reaction to radio-
labeling, C-11-radiolabeled aldehyes, ketones and car-
boxylic acids need to be prepared. In 2000, Perrio-Huard
and coworkers radiolabeled [11C]amines through reduc-
tive amination using [11C]magnesium halide carboxylates,
which was converted directly from [11C]CO2. In 2003,
Van der Meij and coworkers synthesized [11C]acetone
and used it for reductive amination for radiolabeling of
1-phenyl-piperazine [20]. In the meantime, Langstrom
and coworkers established a general approach to
[11C]ketones through Suzuki coupling [21]. Subsequently,
various [11C]ketones were transformed to [11C]amines
through reductive amination. .
Recently, Hooker et al. has reported a simple and di-
rect method for the preparation of C-11 formaldehyde
([11C]HCHO, see general procedure for synthesis) [22].
This opens the way to radiolabel methylated amines
through reductive amination. The advantage of reductive
amination over conventional radiomethylation is that the
reaction can be carried out in aqueous solution. So the
reagents are not necessarily anhydrous. In addition,
base-sensitive functional groups present in the molecule
do not interfere with the reaction, which eliminates pro-
tection /deprotection steps. In this study, we report C-11
labelling of a series of amines through reductive amina-
tion using C-11 formaldehyde in order to demonstrate the
general utility of this method.
2. Results and Discussion
The [11C]CH3I was first converted to [11C]formaldehyde
in the presence of TMAO and directly used for subse-
quent reductive amination without further purification
(see general procedure for 11C-reductive amination of
target compounds) [23]. To test the versatility of the re-
action for C-11 radiolabeling of amines, we used a series
of amine precursors bearing functional groups. Func-
tional groups such as hydroxyl or carboxyl are reactive
toward [11C]CH3I or [11C]CH3OTf under conventional
radiolabeling conditions and ought to be protected first.
Through reductive amination with [11C]formaldehyde,
the aniline derivatives bearing hydroxyl group (1a, 2a, 3a)
were directly radiomethylated in good radiochemical
yield. In contrast to radiomethylation with [11C]CH3I or
[11C]CH3OTf, which must be carried out in strictly anhy-
drous solvents, the reductive animation can be performed
in aqueous solvent like PBS. For compounds that are not
water-soluble, they can be first dissolved in DMF and
diluted with PBS. Thus, the whole process does not re-
quire use of any anhydrous solvents or reagents. The
reductive amination was fast and could be completed
within 5 min. After the reaction, the product can be di-
rectly purified by HPLC with >95% purity. The geome-
try of the hydroxyl group relative to the amino group did
not exert significant impact on the radiochemical yield
(see Purification for compounds 1b - 7b) [24]. Compared
to radiomethylation of 2a and 3a, radiomethylation of 1a
gave a relatively lower yield. This may be due to the fact
that a cyclic hydrogen bond exists between the amino
group and the hydroxyl group in the para-position, deac-
tivating the amino group toward [11C]formaldehyde.
The striking versatility of reductive methylation with
[11C]formaldehyde was also demonstrated by radiome-
thylation of amines bearing a free carboxyl group (4a).
With no protection of the carboxyl group, the amino
group can be readily radiomethylated. While conven-
tional radiomethylation using [11C]CH3I or [11C]CH3OTf
often requires refluxing at temperature over 120˚C, the
reductive amination reaction can be run at relatively low
temperatures. The radiochemical yield was significantly
improved when the reaction temperature was elevated to
90˚C. Interestingly, adding aqueous PBS to the reaction
media dramatically increases the radiochemical yield.
At 90˚C, the radiochemical yield was increased ca. 5-fold
when the reaction was run in DMF-PBS (1:3) versus in
DMF alone.
Radiomethylation using [11C]formaldehyde was found
to be selective for primary amines. The reaction selectiv-
ity for primary amino group was demonstrated in the
reductive methylation reaction of serotonin (5a), a widely
studied neurotransmitter. Previous radiolabeling of sero-
tonin with [11C]CH3I or [11C]CH3OTf required protection
and deprotection of both the hydroxyl and the secondary
amino group. However, under the reductive methylation
condition, the primary amino group was selectively me-
thylated. The radiomethylation reaction was also tem-
perature sensitive. The radiochemical yield was signifi-
cantly improved when the reaction was carried out at
elevated temperature.
Copyright © 2012 SciRes. IJOC
C. Y. WU ET AL.
204
Using this radiomethylation method, we carried out
radiosynthesis of PIB using non-protected radiolabeling
precursor (6a). Current radiolabeling protocol that has
been standardized for clinical trials requires protection of
the 6-hydroxyl group with MOM and subsequent depro-
tection. Without protection, both hydroxyl and amino
groups can be methylated, which requires separation by
HPLC. Using [11C]CH3OTf, a loop method has been
adapted by which the reaction can be run directly in the
loop of HPLC before being injected into the separation
column [19]. However, this loop method is only suitable
for production of a small quantity under strictly anhy-
drous conditions. In contrast, only the amino group of 6a
is radiomethylated by reductive amination, which can be
run in aqueous media with comparable radiochemical
yields. The hydroxyl group remained intact under the
reaction condition [25].
Recently, we reported [11C]MeDAS (7b) as a PET ra-
dioligand for myelin imaging [26]. Compared to previ-
ously reported radiolabeling using [11C]CH3OTf, radio-
synthesis of [11C]MeDAS was also achieved in aqueous
solution through reductive amination in a much shorter
time albeit a relatively lower radiochemical yield.
3. Experimental Section
General procedure for synthesis of [11]CH2O: The
target gas (99.5% Nitrogen and 0.5% oxygen) was
loaded on the cyclotron target and was bombarded by the
cyclotron beam at 40 µA for 5 to 10 min. After that, the
cyclotron-generated 11CO2 was delivered into a three-
neck reaction vessel equipped with water-cooling system
and reduced to [11C]CH3OH by lithium aluminum hy-
dride (LAH, 0.1 M) solution in tetrahydrofuran (1.2 ml).
Then hydriodic acid 57 wt% in water (0.9 ml) was added
into the vessel to generate the [11C]CH3I at 120˚C, which
was concurrently distilled and trapped into a dry 3-ml
conical reaction vial with screw cap which was previ-
ously filled with a mixture of trimethylamine N-oxide
(TMAO, 5 mg) and DMF (200 µL) at –40˚C. Trapping of
[11C]CH3I was monitored by measuring the radioactivity
in the isotope calibrator until the maximal value was at-
tained. Then, the sealed vial was heated to 70˚C and
maintained for 2 min. After that, the reaction vial was
cooled to room temperature by dry ice in 2 min.
General procedure for 11C-reductive amination of
target compounds: All of the mixture in the 3-ml reac-
tion vial was collected and transferred into a dry 1-ml
conical reaction vial with screw cap which was previ-
ously filled with target molecular compound (1 mg), so-
dium cyanoborohydride (5 mg) and sodium phosphate
buffer (0.04 M, pH 7.0, 600 µL). Then, the sealed vial
was heated to 70˚C or 90˚C and maintained for 5 min.
After that, the vial was cooled to room temperature by
dry ice in 2 min. In order to increase the radiochemical
yield of C-11 reductive amination of target compounds,
we assayed the effect of the solvent and the reaction
temperature. We found if we used aqueous sodium
phosphate buffer (0.04 M, pH 7.0, 600 µL) as reaction
solvent, and the reaction temperature was 90˚C, the ra-
diochemical yield was significantly increased (for exam-
ple, see Table 1, entry 4- 11).
Purification for compounds 1b - 3b: The radiolabeled
reaction mixture was directly loaded on to a semi-
preparative HPLC (Phenomenex Luna C18 10 μm, 10 ×
250 mm) column and was eluted with a mobile phase
containing water and acetonitrile (5% for first 2 min,
then a linear gradient from 5% to 95% in 15 min) at a
flow rate of 3 ml/min and UV absorbance at 254 nm The
retention times for compounds 1b, 2b and 3b are 12.82
min, 12.86 min and 12.22 min, respectively. Purification
for compound 4b: The radiolabeled reaction mixture was
directly loaded onto a semi-preparative HPLC (Phe-
nomenex Luna C18 10 μm, 10 × 250 mm) column and
was eluted with a mobile phase containing NH4Cl/HCl
buffer (pH 3.0) and acetonitrile (a linear gradient from 20
to 100% in 20 min) at a flow rate of 3 ml/min and UV
wavelength at 254 nm. The retention time for compound
4b is 10.95 min. Purification for compound 5b: The ra-
diolabeled reaction mixture was directly loaded onto a
semi-preparative HPLC (Phenomenex Luna C18 10 μm,
10 × 250 mm) column and was eluted with a mobile
phase containing water and acetonitrile (5% for first 2
min, then a linear gradient from 5 to 95% in 25 min) at a
flow rate of 3 ml/min and UV absorbance at 254nm The
retention time for compound 5b is 5.68 min.
Purification for compounds 6b and 7b: To the ra-
diolabeled reaction mixture was added 10 ml of water.
The mixture was then passed through a Waters Light
C-18 Sep-Pak cartridge previously conditioned with 10
ml of ethanol followed by 10 ml of water to remove
non-organic impurities. The Sep-Pak cartridge was
washed with another 10 ml of water and dried with a
rapid air bolus. Then, the C-18 Sep-Pak cartridge was
eluted with 1ml of ethanol, and the radiolabeled product
was collected and loaded onto a semi-preparative HPLC
(Phenomenex Luna C18 10 μm, 10 × 250 mm) column
and was eluted with a mobile phase containing water and
acetonitrile (4:6, v/v) at a flow rate of 3 ml/min and UV
wavelength at 365 nm The retention times for 6b and 7b
were 4.83 min and 6.53 min, respectively.
4. Conclusion
In conclusion, reductive amination using [11C]formal-
dehyde proves to be a versatile approach to C-11 radio-
labeling of amines. As demonstrated with different sub-
strates bearing functional groups that are sensitive to
Copyright © 2012 SciRes. IJOC
C. Y. WU ET AL.
Copyright © 2012 SciRes. IJOC
205
Table 1. 11C-Reductive Amination of Target Compoundsa.
11CH 3I11CH2O
11CO2
1) LAH/THF
2) HI, 120oC
TMAO
DMF,70oCPBS,NaCNBH3
70- 90oC
R-NH2
(1a-7a)R-NH-11CH3
(1b-7b)
Entry Precursor Product Solvents
Temp
(˚C)
Reaction
Time (min)
Purity
(%)d
Radiochemical
Yield (%)e
1
DMF-PBS (1:3)70 5 >95 23.7
1a 1b
2
NH2
OH
DMF-PBS (1:3)70 5 >95 31.6
2a 2b
3
DMF-PBS (1:3)70 5 >95 33.3
3a 3b
4 DMF-PBS (1:3)70 5 >95 1.8
5b DMF 90 5 >95 3.0
6 DMF-PBS (1:3)90 5 >95 14.8
4a 4b
7 DMF-PBS (1:3)70 5 >95 2.1
8
DMF-PBS (1:3)90 5 >95 26.5
5a 5b
9b DMF 70 5 >95 1.6
10b DMF 90 5 >95 2.6
11c DMF-PBS (1:1)90 5 >95 7.9
6a 6b
12 DMF-PBS (1:3)70 5 >95 28.7
7a 7b
aConditions: LAH/THF (0.1M, 1.2 ml), HI (57 wt% in H2O, 0.9 ml), TMAO (5 mg), DMF (200 μL), Target compounds 1a - 7a (1 mg), NaCNBH3 (5 mg), PBS
(0.04 M, pH 7.0, 600 µL); bConditions: add DMF (200 µL) for dissolving TMAO firstly, then add DMF (600 µL) for dissolving target compounds 4a and 6a in
the next step; cConditions: add DMF (200 µL) for dissolving TMAO first, then add DMF (200 µL) for dissolving target compound 6a and dilute the mixture
with PBS (0.04 M, pH 7.0, 400 µL); dRadiochemical purity determined by HPLC; eRadiochemical yield is decay-corrected to 11CH2O radioactivity.
[11C]CH3I or [11C]CH3OTf, this method radiolabels only
the amino group, which can be carried out in aqueous
solvents in a short time.
5. Supplementary Material
Detailed experimental procedures for the cold synthesis
of compounds 1b-3b and 7b, and 1H and 13C NMR spec-
tra; HPLC spectra for hot synthesis of compounds 1b -
7b.
6. Acknowledgements
We gratefully acknowledge financial support through
grants from the Clinical and Translational Science Col-
laborative (CTSC) pilot grant, the Coulter-Case Founda-
tion, the Department of Defense, National Multiple Scle-
rosis Society, and NIH/NINDS (R01 NS061837).
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C. Y. WU ET AL.
208
Supplemental Information
1. General Information
All chemicals, unless otherwise stated, were purchased
from Sigma-Aldrich and used without further purifica-
tion. 1HNMR spectra were obtained at 400 MHz on an
Inova 400 NMR system using 5 mm NMR tubes (Wil-
mad 528-PP) in CDCl3 or DMSO-d6 (Aldrich or Cam-
bridge Isotopes) solutions at room temperature. Chemical
shifts are reported as d values relative to internal TMS.
HR-ESIMS were acquired under the electron spray ioni-
zation (ESI) condition. Radiochemical purity was deter-
mined by an Agilent 1100 high-pressure liquid chroma-
tography (HPLC) system equipped with UV and Raytest
gamma count detectors.
2. Procedures and Experimental Data
Preparation of 2-(methylamino)-benzyl alcohol (1b)
and 4-(methylamino)-benzyl alcohol (3b)
2-(Methylamino)-benzyl alcohol (1b)
To a 25 ml two-neck flask fitted with a magnetic stir
bar were added 1a (1.5 mmol, 190 mg), zeolite NaY (1
mg, dried at 70˚C overnight) and 5 ml of anhydrous
DMC. The resulting mixture was then stirred at room
temperature for 5 min followed by refluxing at 90˚C for
24 hrs. Once the reaction was completed (monitored by
TLC), the mixture was filtered and washed with CH2Cl2.
The solvent was combined and removed by rotary
evaporation. The crude product was purified by flash
chromatography (Hexane/Ethyl acetate = 3:1, v/v) to
yield 1b (150 mg, 1.1 mmol, 73%). 1b: 1H NMR (400
MHz, CDCl3): δ 7.27-7.24 (m, 1H), 7.08 - 7.05 (m, 1H),
6.69-6.65 (m, 2H), 4.61 (S, 2H), 2.86 (S, 3H). HRMS
(ESI) calcd. for C8H11NO (m/z M + H+):138.09134,
Found 138.09135. HPLC (Phenomenex Luna C18 5 μm,
10 × 250 mm, water and acetonitrile (5% for first 2 min,
then a linear gradient from 5 to 95% in 15 min, flow rate:
3 ml/min, UV 254nm), retention time is 12.82 min.
4-(Methylamino)-benzyl alcohol (3b)
To a 25 ml two-neck flask fitted with a magnetic stir
bar were added 3a (1.5 mmol, 190 mg), zeolite NaY (1
mg, dried at 70˚C overnight) and 5 ml of anhydrous
DMC. The resulting mixture was then stirred at room
temperature for 5 min followed by refluxing at 90˚C for
24 hrs. Once the reaction was completed (monitored by
TLC), the mixture was filtered and washed with CH2Cl2.
The solvent was combined and removed by rotary
evaporation. The crude product was purified by flash
chromatography (Hexane/Ethyl acetate = 3:1, v/v) to
yield 3b (72 mg, 0.53 mmol, 35%). 3b: 1H NMR (400
MHz, CDCl3): δ 7.24-7.17 (m, 2H), 6.61-6.57 (m, 2H),
4.54 (S, 2H), 2.83 (S, 3H). HRMS (ESI) calcd. for
C8H11NO (m/z M + H+):138.09134, Found 138.09135.
HPLC (Phenomenex Luna C18 5μm, 10 × 250 mm, wa-
ter and acetonitrile (5% for first 2 min, then a linear gra-
dient from 5 to 95% in 15 min, flow rate: 3 ml/min, UV
254 nm), retention time is 12.22 min.
Preparation of 3-(methyl am ino)-b enz yl alcohol (2b)
To a suspension of 2a (1.0 mmol, 123 mg) and for-
maldehyde (1.1 mmol, 33.0 mg) was added 2-propanol
(1.5 ml). After 3 hrs of continuous agitation at room
temperature, NaBH4 (1.5 mmol, 60 mg) was added and
the reaction mixture was allowed to stir overnight. Once
the reaction was complete (monitored by TLC), the mix-
ture was filtered and washed with CH2Cl2. The solvent
was removed by rotary evaporation. The crude product
was purified by chromatography (Hexane/Ethyl acetate =
2:1, v/v) to yield product 2b (40 mg, 0.29 mmol, 29%).
1HNMR(400 MHz, CDCl3): δ 7.27 - 7.24 (m, 1H), 7.08 -
7.05 (m, 1H), 6.69 - 6.65 (m, 2H), 4.61 (S, 2H), 2.86 (S,
3H). HRMS (ESI) calcd. for C8H11NO (m/z M +
H+):138.09134, Found 138.09134. HPLC (Phenomenex
Luna C18 5 μm, 10 × 250 mm, water and acetonitrile
(5% for first 2 min, then a linear gradient from 5 to 95%
in 15 min, flow rate: 3 ml/min, UV 254 nm), retention
time is 12.86 min.
Copyright © 2012 SciRes. IJOC
C. Y. WU ET AL. 209
Preparation of (E)-N-methyl-4-(4-nitrostyryl)aniline (7b)
Preparation of tert-butyl (4-((diethoxyphosphoryl)
methyl)phenyl)carbamate (2).
To a 100 ml round bottom flask fitted with a magnetic
stir bar were added diethyl 4-aminobenzylphosphonate (1,
2.500 g, 10.28 mmol) and di-tert-butyl dicarbonate
(2.240 g, 10.26 mmol) in THF (15 ml) and water (6 ml).
The reaction was stirred at room temperature overnight.
THF was removed in vacuo and the remaining residue
was suspended in ethyl acetate and water. The aqueous
layer was extracted three times with ethyl acetate. The
organic layers were combined and washed twice with
water and once with brine. The organic layer was dried
over MgSO4, filtered, and concentrated to give 2 as a
white powder (3.393 g, 96%) and was used without fur-
ther purification. 1H-NMR (CDCl3, 400 MHz): δ 7.31 (d,
J = 8.4 Hz, 2H), 7.21 (m, 2H), 6.47 (br s, 1H), 4.00 (m,
4H), 3.09 (d, 2JHP = 21.2 Hz, 2H), 1.51 (s, 9H), 1.24 (td,
3JHH = 6.8, 4JHP = 0.4 Hz, 6H).
Preparation of tert-butyl (4-((diethoxyphosphoryl)
methyl)phenyl)(methyl)carbamate (3).
Compound 2 (503 mg, 1.46 mmol) was added to an
oven dried 25 ml round bottom flask fitted with a mag-
netic stir. The flask was purged with argon then dry THF
(3.0 ml) was added to dissolve 2. The flask was cooled to
0˚C. NaH (93.0 mg, 2.33 mmol, 60% dispersion in min-
eral oil) was placed in a 2 ml vial and washed with hex-
anes (1.5 ml × 3). The NaH was suspended in dry THF
(6.0 ml) and added to the solution of 2 under positive
argon pressure. MeI (200 µL, 2.91 mmol, 2.28 g/ml) was
added to the reaction mixture under positive argon pres-
sure. The reaction was stirred at 0˚C under argon then
slowly warmed to room temperature and stirred over-
night. The reaction was quenched with water. THF was
removed in vacuo. The remaining residue was suspended
in ethyl acetate and water and the aqueous layer was ex-
tracted three times with ethyl acetate. The organic layers
were combined and washed twice with water and once
with brine. The organic layer was dried over MgSO4,
filtered, and concentrated to give 3 as a yellow oil (451
mg, 86%) and was used without further purification.
1H-NMR (CDCl3, 400 MHz): δ 7.25 (m, 2H), 7.16 (d, J =
8.4 Hz, 2H), 4.01 (m, 4H), 3.23 (s, 3H), 3.12, 2JHP = 21.6
Hz, 2H), 1.43 (s, 9H), 1.24 (t, 3JHH = 7.0 Hz, 6H).
Preparation of (E)-tert-butyl methyl(4-(4-nitrostyryl)
phenyl)carbamate (4)
An oven dried 50 ml round bottom flask fitted with a
magnetic stir bar was purged with argon and 3 (957 mg,
Copyright © 2012 SciRes. IJOC
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210
2.68 mmol) was added in dry DMF (3.0 ml). In a 2 ml
vial, NaH (377 mg, 9.42 mmol, 60% dispersion in min-
eral oil) was washed with hexanes (1.5 ml × 3) then
added to the solution of 3 in 9.0 ml DMF under positive
argon pressure. The reaction was stirred under argon at
room temperature for 20 minutes. 4-Nitrobenzaldehyde
(383 mg, 2.53 mmol) was added to the reaction mixture
in dry DMF (6.0 ml) under positive argon pressure. The
reaction was stirred overnight under argon at room tem-
perature. The reaction was quenched with water and the
aqueous phase was extracted three times with ethyl ace-
tate. The organic layers were combined and washed
twice with water and once with brine. The organic layer
was dried over MgSO4, filtered, and concentrated. The
crude red-orange solid was recrystallized in hot EtOH to
give 4 as red crystals (529 mg, 24%). 1H-NMR (CDCl3,
400 MHz) : δ 8.22 (ddd, J = 9.6, 4.4, 2.4 Hz, 2H), 7.63
(ddd, J = 9.2, 4.4, 2.4 Hz, 2H), 7.51 (ddd, J = 9.6, 4.8,
2.8 Hz, 2H), 7.29 (m, 2H), 7.24 (d, J = 16.4 Hz, 1H),
7.09 (d, J = 16.4 Hz, 1H), 3.29 (s, 3H), 1.48 (s, 9H).
Preparation of (E)-N-methyl-4-(4-nitrostyryl)aniline
(5)
Compound 4 (522 mg, 1.47 mmol) was added to a 10
ml round bottom flask fitted with a magnetic stir bar and
dissolved in neat TFA (6.4 ml, 83 mmol, 1.48 g/ml). The
reaction was stirred at room temperature for 2 h. The
reaction was quenched with 4M NaOH (~40 ml) and the
aqueous solution was extracted with ethyl acetate three
times. The organic layers were combined and washed
twice with water and once with brine. The organic layer
was dried over MgSO4, filtered, and concentrated to give
5 as a red solid (365 mg, 97%) and was used without
further purification. 1H-NMR (CDCl3, 400 MHz): δ 8.18
(m, 2H), 7.56 (m, 2H), 7.41 (m, 2H), 7.20 (d, J = 16.2 Hz,
1H), 6.92 (d, J = 16.2 Hz, 1H), 6.61 (m, 2H), 3.97 (br s,
1H), 2.89 (s, 3H).
Preparation of (E)-4-(4-aminostyryl)-N-methylaniline
(7b)
Compound 6 (355 mg, 1.40 mmol) was added to a 250
ml round bottom flask fitted with a magnetic stir bar in
ethyl acetate (60 ml) and ethanol (30 ml). Tin (II) chlo-
ride (4.72 g, 24.9 mmol) was added to the solution of 6.
A water condenser was attached and the reaction was
stirred at 70˚C overnight. The solvent was removed in
vacuo. The residue was suspended in 4 M NaOH to pre-
cipitate the product. The mixture was filtered through a
fine glass frit and washed several times with 4 M NaOH
followed by water until the pH of the wash water was 7.
Then, the solid on the frit was washed with ethyl acetate
into a clean flask. That solution was dried over MgSO4,
filtered, and concentrated to give the crude product. The
crude product was dissolved in CH2Cl2 and silica gel.
The solvent was carefully removed leaving the crude
product absorbed onto silica gel. This solid was added to
the top of a silica column and purified by flask chroma-
tography with Et2O/Et3N (49:1). Concentration in vacuo
gave 7b as a light orange solid (224 mg, 72%). 1H-NMR
(CDCl3, 400 MHz): δ 7.36 (ddd, J = 9.2, 4.8, 2.8 Hz, 2H),
7.32 (ddd, J = 9.2, 4.8, 2.8 Hz, 2H), 6.90 (d, J = 16.2 Hz,
1H), 6.85 (d, J = 16.2 Hz, 1H), 6.67 (ddd, J = 9.2, 4.8,
2.8 Hz, 2H), 6.61 (ddd, J = 9.2, 4.8, 2.8 Hz, 2H), 3.72 (br
s, NHCH3 + NH2, 3H), 2.86 (s, 3H). HR-ESIMS: m/z
calcd for C15H16N2 (M + H+), 225.1386; found, 225.1385.
HPLC (Phenomenex Luna C18 5μm, 10 × 250mm, wa-
ter : acetonitrile = 4:6 (v/v), flow rate: 3ml/min, UV 365
nm), retention time is 5.63 min.
General procedure for synthesis of 11CH2O
The cyclotron-made [11C]carbon dioxide was trans-
ferred into a three-neck reaction vessel with wa-
ter-cooling system and reduced to 11CH3OH by lithium
aluminum hydride (LAH, 0.1M solution in THF). Once
the reaction mixture is dry enough, hydriodic acid (0.9
ml, 57 wt% in water) was added into the vessel to gener-
ate labeled methyl iodide 11CH3I at 120˚C. 11CH 3I was
concurrently distilled and trapped in a dry 3-ml conical
reaction vial with a screw cap which was previously
filled with a mixture of trimethylamine N-oxide (TMAO,
5 mg) and DMF (200 µL) at –40˚C. Trapping of [11C]
methyl iodide was monitored by measuring the activity
in an isotope calibrator until the maximum value was
attained. Then the reaction mixture was sealed and
heated at 70˚C for 2 minutes in a heating block. After
cooling to room temperature in an ice bath, 11CH2O was
ready for use.
Synthesis of 11C-1b
11C-1b
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C. Y. WU ET AL. 211
H11CHO generated in the above reaction vial was trans-
ferred into a dry 1-ml conical reaction vial with screw cap
which was previously filled with (2-aminophenyl)metha-
nol (1 mg), sodium cyanoborohydride (5 mg) and sodium
phosphate buffer (0.04 M, pH 7.0, 600 µL). The sealed
vial was then heated to 70˚C and maintained for 5 min.
After cooling to room temperature, the radiolabeled reac-
tion mixture was directly loaded onto a semi-preparative
HPLC (Phenomenex Luna C18, 10μm, 10 × 250 mm)
column and was eluted with a mobile phase containing
water and acetonitrile (5% for first 2 min, then a linear
gradient from 5 to 95% in 15 min) at a flow rate of 3
ml/min and a UV absorbance at 254 nm. The retention
time of 11C-1b is 12.82 min. Identification of 11C-1b and
radiochemical purity were verified by co-injection with
the non labeled cold standard of 1b which has the same
retention time on the UV and radioactive chromatograms.
The radiochemical purity of 11C-1b was >95% after
HPLC purification. The decay-corrected radiochemical
yield of [11C]1b obtained after HPLC purification was
23.7% based on the radioactivity of [11C]methyl iodide
trapped.
Synthesis of 11C-2b
11C-2b
H11CHO generated in the above reaction vial was trans-
ferred into a dry 1-ml conical reaction vial with screw cap
which was previously filled with (3-aminophenyl)metha-
nol (1 mg), sodium cyanoborohydride (5 mg) and sodium
phosphate buffer (0.04 M, pH 7.0, 600 µL). The sealed
vial was then heated to 90˚C and maintained for 5 min.
After cooling to room temperature, the radiolabeled reac-
tion mixture was directly loaded on to a preparative
HPLC (Phenomenex Luna C18, 10μm, 10 × 250 mm)
column and was eluted with a mobile phase containing
water and acetonitrile (5% for first 2 min, then a linear
gradient from 5 to 95% in 15 min) at a flow rate of 3
ml/min and a UV wavelength at 254 nm. The retention
time is 12.86 min. Identification of 11C-2b and radio-
chemical purity were verified by co-injection with the
non labeled cold standard of 2b which has the same re-
tention time on the UV and radioactive chromatograms.
The radiochemical purity of 11C-2b was >95% after
HPLC purification. The decay-corrected radiochemical
yield of [11C]2b obtained after HPLC purification was
31.6% base on the radioactivity of [11C]methyl iodide
trapped.
Synthesis of 11C-3b
11C-3b
H11CHO generated in the above reaction vial was trans-
ferred into a dry 1-ml conical reaction vial with screw cap
which was previously filled with (4-aminophenyl)
methanol (1 mg), sodium cyanoborohydride (5 mg) and
sodium phosphate buffer (0.04 M, pH 7.0, 600 µL). The
sealed vial was then heated to 90˚C and maintained for 5
min. After cooling to room temperature, the radiolabeled
reaction mixture was directly loaded on to a preparative
HPLC (Phenomenex Luna C18, 10 μm, 10 × 250 mm)
column and was eluted with a mobile phase containing
water and acetonitrile (5% for first 2 min, then a linear
gradient from 5 to 95% in 15 min) at a flow rate of 3
ml/min and a UV wavelength at 254 nm. The retention
time is 12.22 min. Identification of 11C-1b and radio-
chemical purity were verified by co-injection with the
non labeled cold standard of 3b which has the same re-
tention time on the UV and radioactive chromatograms.
The radiochemical purity of 11C-3b was >95% after
HPLC purification. The decay-corrected radiochemical
yield of [11C]3b obtained after HPLC purification was
33.3% base on the radioactivity of [11C]methyl iodide
trapped.
Synthesis of 11C-4b
11C-4b
H11CHO generated in the above reaction vial was
transferred into a dry 1 ml conical reaction vial with
screw cap which was previously filled with 4-amino-
benzoic acid (1 mg), sodium cyanoborohydride (5 mg)
and sodium phosphate buffer (0.04 M, pH 7.0, 600 µL).
The sealed vial was then heated to 70˚C and maintained
for 5 min. After cooling to room temperature, the radio-
labeled reaction mixture was directly loaded on to a
preparative HPLC (Phenomenex Luna C18, 10μm, 10 ×
250 mm) column and was eluted with a mobile phase
containing NH4Cl/HCl buffer (pH 3.0) and acetonitrile (a
linear gradient from 20 to 100% in 20 min).at a flow rate
of 3 ml/min and a UV wavelength at 254 nm. The reten-
tion time for compound 4b is 10.95 min. Identification of
11C-4b and radiochemical purity was verified by
co-injection with the non labeled cold standard of 4b
which has the same retention time on the UV and radio-
active chromatograms. The radiochemical purity of
11C-4b was >95% after HPLC purification. The de-
cay-corrected radiochemical yield of [11C]4b obtained
after HPLC purification was 1.8% base on the activity of
Copyright © 2012 SciRes. IJOC
C. Y. WU ET AL.
212
[11C]methyl iodide trapped. In order to increase the ra-
diolabelling yield of [11C]4b, we used either DMF 600µL
or sodium phosphate buffer (0.04 M, pH 7.0, 600 µL) as
reaction solvent, the reaction temperature was set to 90oC
and maintained for 5 min. The radiochemical yield of
3.0% and 14.8% can be attained based on the radioactiv-
ity of [11C]methyl iodide trapped.
Synthesis of 11C-5b
11C-5b
H11CHO generated in the above reaction vial was
transferred into a dry 1-ml conical reaction vial with
screw cap which was previously filled with 3-(2-amino-
ethyl)-1H-indol-5-ol (1 mg), sodium cyanoborohydride
(5 mg) and sodium phosphate buffer (0.04 M, pH 7.0,
600 µL). The sealed vial was then heated to 70˚C and
maintained for 5 min. After cooling to room temperature,
the radiolabeled reaction mixture was directly loaded on
to a preparative HPLC (Phenomenex Luna C18, 10μm,
10 × 250 mm) column and was eluted with a mobile
phase containing water and acetonitrile (5% for first 2
min, then a linear gradient from 5 to 95% in 25 min).at a
flow rate of 3 ml/min and a UV wavelength at 254 nm.
The retention time is 5.68 min. Identification of 11C-5b
and radiochemical purity was verified by co-injection
with the non labeled cold standard of 5b which has the
same retention time on the UV and radioactive chroma-
tograms. The radiochemical purity of 11C-5b was >95%
after HPLC purification. The decay-corrected radio-
chemical yield of [11C]5b obtained after HPLC purifica-
tion was 2.1% base on the activity of [11C]methyl iodide
trapped. In order to increase the radiolabelling yield of
[11C]5b, we set the reaction temperature to 90˚C and
maintained for 5 min. The radiochemical yield increased
to 26.5% based on the radioactivity of [11C]methyl iodide
trapped.
Synthesis of 11C-6b
11C-6b
H11CHO generated in the above reaction vial was trans-
ferred into a dry 1-ml conical reaction vial with screw cap
which was previously filled with 2-(4-aminophenyl)
benzo[d]thiazol-6-ol (1 mg), sodium cyanoborohydride
(5 mg) and sodium phosphate buffer (0.04 M, pH 7.0,
600 µL). The sealed vial was then heated to 70˚C and
maintained for 5 min. After cooling to room temperature,
the reaction mixture was diluted with water and was
passed through a Waters C-18 Sep-Pak cartridge previ-
ous conditioned with ethanol then water to remove the
non-organic impurities. The Sep-Pak cartridge was
washed with 10 ml of water and dried with a rapid air
bolus, and the radiolabeled product was eluted with
ethanol and was loaded onto a preparative HPLC (Phe-
nomenex Luna C18, 10 µ C18 10 × 250 mm) column and
was eluted with a mobile phase containing water and
acetonitrile (4:6 v/v) at a flow rate of 3 ml/min and a UV
wavelength at 365 nm. The retention time is 4.83 min.
Identification of 11C-6b is verified by coinjection with
the non labeled cold standard 6b, which has the same
retention time on the UV and radioactive chromatograms.
The radiochemical purity of 11C-6b af ter HPLC prepara-
tion is greater than 95%. The radiochemical yield was
approximately 10%.
Synthesis of 11C-7b
11C-7b
H11CHO generated in the above reaction vial was
transferred into a dry 1-ml conical reaction vial with
screw cap which was previously filled with (E)-4,4'-
(ethene-1,2-diyl)dianiline (1 mg), sodium cyanoboro-
hydride (5 mg) and sodium phosphate buffer (0.04 M,
pH 7.0, 600 µL). The sealed vial was then heated to 70oC
and maintained for 5 min. After cooling to room tem-
perature, the reaction mixture was diluted with water and
was passed through a Waters C-18 Sep-Pak cartridge
previous conditioned with ethanol then water to remove
the non-organic impurities. The Sep-Pak cartridge was
washed with 10 ml of water and dried with a rapid air
bolus, and the radiolabeled product was eluted with
ethanol and was loaded onto a preparative HPLC (Phe-
nomenex Luna C18, 10µ C18 10 × 250 mm) column and
was eluted with a mobile phase containing water and
acetonitrile (4:6 v/v) at a flow rate of 3 ml/min and a UV
wavelength at 365 nm. The retention time is 6.53 min.
Identification of 11C-7b and radiochemical purity was
verified by co-injection with the non labeled cold stan-
dard of 7b which has the same retention time on the UV
and radioactive chromatograms. The radiochemical pu-
rity of 11C-7b was >95% after HPLC purification. The
decay-corrected radiochemical yield of [11C]7b obtained
after HPLC purification was 28.7% based on the activity
of [11C]methyl iodide trapped.
3. 1HNMR Spectra of Novel Compounds
2-(Methylamino)-benzyl alcohol (1b)
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213
3-(Methylamino)-benzyl alcohol (2b)
C. Y. WU ET AL.
214
4-(Methylamino)-benzyl alcohol (3b)
(E)-4-(4-aminostyryl)-N-methylaniline (7b)
ppm (t1
)
1.02.03.04.05.06.07.08.0
200
0
150
0
100
0
500
0
3.95
2.00
4.01
2.92
3.00
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4. HRMS(ESI) Spectra of Novel Compounds
2-(Methylamino)-benzyl alcohol (1b)
3-(Methylamino)-benzyl alcohol (2b)
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216
4-(Methylamino)-benzyl alcohol (3b)
(E)-4-(4-aminostyryl)-N-methylaniline (7b)
222 224226 228
m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
225.13850
224.13078 226.14198
227.14542
223.12308 228.33698
225.13863
226.14198
227.14534 229.15205
NL:
5.97E6
Norbert-O-0702
001#471 RT:
FTMS + p ESI
[50.00-1000.
NL:
8.43E5
C15 H17N2:
C15 H17N2
c(gss, s/p:40
R: 150000 Re
M+H
+
Sample
M+H
+
The or e ti ca l
08-MMB-
3.64 AV: 1 F:
Full ms
00]
)(Val) Chrg1
s.Pwr. @FWHM
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5. HPLC Spectra for both Cold and Hot Syntheses
11C-1b
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218
11C-2b
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11C-3b
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220
11C-4b
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C. Y. WU ET AL. 221
11C-5b
Copyright © 2012 SciRes. IJOC
C. Y. WU ET AL.
222
11C-6b
Copyright © 2012 SciRes. IJOC
C. Y. WU ET AL.
Copyright © 2012 SciRes. IJOC
223
11C-7b