Synthesis of 2,6-difluoro-N-(3-[11C]methoxy-1H-pyrazolo[3,4-b] pyridine-5-yl)-3-(propylsulfonamidio)benzamide as a new potential PET agent for imaging of B-RafV600E in cancers

Article abstract of DOI:10.1016/j.bmcl.2012.12.027

The authentic standard 2,6-difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b] pyridine-5-yl)-3-(propylsulfonamidio)benzamide was synthesized from 2,6-difluorobenzoic acid and 3-amino-5-hydroxypyrazole in 9 steps with 1% overall chemical yield. Direct desmethylation

Full text of DOI:10.1016/j.bmcl.2012.12.027

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1017–1021
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 2,6-difluoro-N-(3-[11C]methoxy-1H-pyrazolo[3,4-b]
pyridine-5-yl)-3-(propylsulfonamidio)benzamide as a new potential
PET agent for imaging of B-RafV600E in cancers
Min Wang a, Mingzhang Gao a, Kathy D. Miller b, Qi-Huang Zheng a,
⇑
a Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
b Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The authentic standard 2,6-difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfo-
namidio)benzamide was synthesized from 2,6-difluorobenzoic acid and 3-amino-5-hydroxypyrazole in
9 steps with 1% overall chemical yield. Direct desmethylation of the reference standard with TMSCl/
NaI gave the precursor 2,6-difluoro-N-(3-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonam-
idio)benzamide for radiolabeling in 70% yield. The target tracer 2,6-difluoro-N-(3-[11C]methoxy-1H-pyr-
azolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide was prepared from the precursor with
Received 16 November 2012
Revised 27 November 2012
Accepted 10 December 2012
Available online 20 December 2012
Keywords:
[
11C]CH3OTf through O-[11C]methylation and isolated by HPLC combined with SPE in 40–50% decay cor-
2,6-Difluoro-N-(3-[11C]methoxy-1H-
pyrazolo[3,4-b]pyridine-5-yl)-3-
(propylsulfonamidio)benzamide ([11C]9)
B-RafV600E
rected radiochemical yields with 370–740 GBq/
l
mol specific activity at end of bombardment (EOB).
Ó 2012 Elsevier Ltd. All rights reserved.
Radiosynthesis
Automation
Positron emission tomography (PET)
Cancer imaging
The mitogen-activated protein kinase (MAPK)/extracellular sig-
nal-regulated kinase (ERK) signaling pathway transduces signals
from cell surface receptors to the nucleus leading to cell prolifera-
tion, differentiation, and survival.1–4 The serine/threonine-protein
kinase (Raf) family contains A-Raf, B-Raf and C-Raf (Raf-1), and
their biochemical potencies to phosphorylate and activate MAP/
ERK kinase (MEK) are B-Raf > C-Raf >> A-Raf.5 The V600E mutation
due to the substitution of valine with glutamic acid at amino acid
(aa) residue 600 of B-Raf kinase (B-RafV600E) results in constitutive
activation of the Ras (a G-protein)/B-Raf/MEK/ERK (MAPK) signal-
ing pathway and is found in approximately 7% of all cancers
including melanoma, papillary thryroid cancer, colorectal cancer,
cholangiocarcinoma, and ovarine cancer.1–5 B-RafV600E correlates
with increased malignancy and decreased response to chemother-
apy, and thus has emerged as an important biomarker for diagno-
sis, prognosis and therapeutic guidance for human cancers.2
B-RafV600E is a highly attractive target for cancer therapy, and
several small-molecule protein kinase inhibitors selective for
B-RafV600E including RAF265 (Chiron/Novartis), XL281/BMS-908662
(Exelixis/BMS), GSK2118436 (GlaxoSmithKline), and PLX4032
(Plexxikon/Roche) are currently in clinical trials.1–5 Recently, a
novel series of pyrazolopyridine inhibitors of B-RafV600E has been
developed.5 The representative compound 2,6-difluoro-N-(3-
methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)
benzamide (9, IC50 4.8 nM for B-RafV600E) is a selective, orally
bioavailable, and efficacious inhibitor and has been selected for
further preclinical evaluation.5 B-RafV600E is an attractive target
for molecular imaging of cancer as well. However, so far no specific
and selective cancer B-RafV600E imaging agent to guide chemother-
apy is reported, and the therapeutic efficacy of PLX4032 for the
treatment of malignant melanoma patients was observed through
the reductions in 2-[18F]fluoro-2-deoxyglucose ([18F]FDG) uptake
on biomedical imaging technique positron emission tomography
(PET) scanning, in which [18F]FDG is the only PET cancer imaging
agent for glucose metabolism used clinically at this point in time.6
We are interested in the development of new PET imaging agents
for cancer detection, diagnosis and image-guided therapy, and
here we present the design and synthesis of 2,6-difluoro-N-
(3-[11C]methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfo-
namidio)benzamide ([11C]9) as
a new specific and selective
potential imaging agent for PET to image B-RafV600E in cancers,
for the first time.
The reference standard 9 was synthesized via amide coupling of
2,6-difluoro-3-(propylsulfonamido)benzoic acid (5) with 3-meth-
oxy-1H-pyrazolo[3,4-b]pyridine-5-amine (8) using the modifica-
tions of the literature method.5,7
⇑
Corresponding author. Tel.: +1 317 278 4671.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.027

1018
M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1017–1021
F
F
F
fuming HNO3
conc. H2SO4
H2, 10% Pd-C
EtOH
conc. H2SO4
MeOH
HOOC
H3COOC
H3COOC
NO2
F
F
F
2, 98%
1, 71%
F
O
S
O
F
1.0 N NaOH
THF-MeOH
propane-1-sulfonyl chloride, Et3N
CH2Cl2
NH2
H3COOC
N
S
H3COOC
F
O
O
F
4, 75%
3, 93%
F
O
O
S
HOOC
N
H
F
5, 80%
Scheme 1. Synthesis of the key intermediate 5.
NO2
N
NH2
NH2
sodium nitromalonaldehyde
MeOH, DIAD
PPh3, CH2Cl2
OH
HN
N
HN
H2O
HN
N
N
OCH3
OCH3
7, 39%
6, 11%
NH2
N
H2, 10% Pd-C
EtOAc-MeOH
HN
N
OCH3
8, 87%
Scheme 2. Synthesis of the key intermediate 8.
As indicated in Scheme 1, the key intermediate 5 was synthe-
sized starting from low cost and commercially available 2,6-diflu-
orobenzoic acid. Benzoic acid was esterified to methyl benzoate 1
by treatment with MeOH in the presence of concentrated H2SO4 as
catalyst, in 71% yield. Nitration of methyl benzoate 1 with fuming
HNO3 in concentrated H2SO4 afforded nitrobenzoate 2 in 98%
yield.8 Reduction of the nitro group was performed by catalytic
hydrogenation of 2 with 10% Pd/C in EtOH to give amino-benzoate
3 in 93% yield. Bis-sulfonamide 4 was obtained by treatment ani-
line 3 with propane-1-sulfonyl chloride in the presence of Et3N
in CH2Cl2, in 75% yield. Concurrent hydrolysis of the ester and
one of sulfonamides was achieved with aqueous NaOH in a mix-
ture of THF and MeOH to provide 5 in 80% yield.
As depicted in Scheme 2, the preparation of another key inter-
mediate 8 began with commercial readily available 3-amino-5-
hydroxypyrazole. Methoxyl-substituted aminopyrazole 6 was
obtained in one-step without protecting and deprotecting by
treatment hydroxyaminopyrazole with MeOH in the presence of
triphenylphosphine and diidopropylazodicarboxylate (DIAD) in
11% yield.9,10 Condensation of amniopyrazole 6 with sodium
nitromalonaldehyde monohydrate in water produced the fused
2-ring heterocyclic compound 7 in 39% yield. Reduction of the nitro
group was performed by catalytic hydrogenation of 7 with 10% Pd/
C in EtOH to give 8 in 87% yield.
a multiple-step short synthesis route to synthesize the precursor.
The different commonly used desmethylating agents including
HBr, BBr3, AlCl3/EtSH, LiBr and chlorotrimethylsilane (TMSCl)/NaI
were tested for the desmethylation reaction, and we finally se-
lected TMSCl/NaI for this reaction, which provided the best result.
The desmethylation of 9 with TMSCl/NaI in acetonitrile afforded
the precursor 10 in 70% yield.11,12
The overall chemical yield for the standard 9 in 9 steps was 1%.
The extremely low yield was because of one-step synthesis of com-
pound 6 in only 11% yield. The way to improve the yield would be
using a 3-step synthetic approach including protecting, O-methyl-
ation and deprotecting reactions to synthesize 6. However, it
would require more materials and labors compared to a low-yield
one-step direct and selective O-methylation.
Radiosynthesis of the target tracer
[
11C]9 is indicated in
Scheme 4. The hydroxyl precursor 10 was labeled by [11C]methyl
triflate ([11C]CH3OTf)13,14 through O-[11C]methylation15–17 in ace-
tonitrile at 80 °C under basic condition (2 N NaOH) and isolated
by a semi-preparative high performance liquid chromatography
(HPLC) with a Prodigy C-18 column from Phenomenex and a so-
lid-phase extraction (SPE) with a disposable C-18 Plus Sep-Pak car-
tridge from Waters (a second purification or isolation process)18–20
to produce the corresponding pure radiolabeled compound [11C]9
in 40–50% radiochemical yield, decay corrected to end of bombard-
ment (EOB), based on [11C]CO2. Addition of NaHCO3 to quench the
radiolabeling reaction and to dilute the radiolabeling mixture prior
to the injection onto the semi-preparative HPLC column for purifi-
cation gave better separation of [11C]9 from its hydroxyl precursor
As shown in Scheme 3, amide coupling of 5 and 8 in the pres-
ence of N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide (EDCI)
and hydroxybenzotriazole (HOBt) in DMF provided the standard
9 in 83% yield. In an effort to obtain the precursor for 11C-labeling,
we focused on desmethylation of 9, because one-step direct des-
methylation to prepare the precursor would be better than using
10.18–21 The radiosynthesis was performed in
automated multi-purpose 11C-radiosynthesis module, allowing
a home-built

M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1017–1021
1019
F
O
O
NH2
NH
S
F
N
N
N
O
O
H
EDCI, HOBt
DMF
O
F
S
+
HN
HOOC
N
H
HN
N
F
5
N
OCH3
OCH3
9, 83%
8
F
O
O
S
NH
N
N
TMSCl, NaI
CH3CN
H
O
F
HN
N
OH
10, 70%
Scheme 3. Synthesis of the reference standard 9 and precursor 10.
F
F
O
O
O
O
NH
S
NH
S
[
11C]CH3OTf, CH3CN
2 N NaOH, 80 °C, 3 min
N
N
N
N
H
H
O
F
O
F
HN
HN
N
N
O11CH3
OH
10
[
11C]9, 40-50%
Scheme 4. Synthesis of the target tracer [11C]9.
measurement of specific radioactivity during synthesis.22–24 This
11C-radiosynthesis module includes the overall design of the
reaction, purification and reformulation capabilities of the proto-
has been built, featuring the measurement of specific activity
by the on-the-fly technique. The radiosynthesis employed
O-[11C]methylation radiolabeling on oxygen position of the
hydroxyl precursor. Radiolabeling procedures incorporated effi-
ciently with the most commonly used [11C]methylating agent,
type system. In addition, 11C-tracer specific activity (GBq/
lmol at
EOB) can be automatically determined prior to product delivery
for compounds purified by the HPLC-portion of the system. Briefly,
analysis of the chromatographic data utilized PeakSimple software
(SRI Instruments, Las Vegas, NV). Immediately following elution of
the product peak, the chromatographic data are exported to
PeakSimple readable files, and the area of the radioactivity peak
is converted to GBq at EOB by comparison to a reference calibra-
tion curve previously constructed using the same detector, loop
and flow rate. The mass peak from the UV chromatogram (without
decay correction) is similarly compared to a standard curve made
at the same UV wavelength, mobile phase and flow rate. Simple
division of the total EOB radioactivity peak (in GBq) by the total
[
[
11C]CH3OTf, which was produced by gas-phase production of
11C]methyl bromide ([11C]CH3Br) from our laboratory.14 The
target tracer was isolated and purified by a semi-preparative HPLC
combined with SPE procedure in high radiochemical yields, short
overall synthesis time, and high specific activity. These chemistry
results combined with the reported in vitro and in vivo biological
data of pyrazolopyridine inhibitors5 encourage further in vivo
biological evaluation, preclinical and clinical PET studies of
new radiolabeled pyrazolopyridine inhibitors as cancer B-RafV600E
imaging agents in animals and humans.
mass peak (in nmoles) gives specific activity at EOB in GBq/lmol.
The overall synthesis, purification and reformulation time was
Acknowledgments
30–40 min from EOB. The specific radioactivity was in a range of
370–740 GBq/lmol at EOB. The specific activity can also be mea-
This work was partially supported by the Breast Cancer
Research Foundation. 1H NMR and 13C NMR spectra were recorded
on a Bruker Avance II 500 MHz NMR spectrometer in the Depart-
ment of Chemistry and Chemical Biology at Indiana University
Purdue University Indianapolis (IUPUI), which is supported by a
NSF-MRI Grant CHE-0619254.
sured by analytical HPLC, which is consistent with the on-the-fly
technique to determine the specific activity by semi-preparative
HPLC. Chemical purity and radiochemical purity were determined
by analytical HPLC.25 The chemical purity of the precursor 10 and
reference standard 9 was >97%. The radiochemical purity of the
target tracer [11C]9 was >99% determined by radio-HPLC through
-ray (PIN diode) flow detector, and the chemical purity of [11C]9
References and notes
c
was >93% determined by reverse-phase HPLC through UV flow
detector. A C-18 Plus Sep-Pak cartridge was used to significantly
improve the chemical purity of the tracer solution.18–20,25 The
chemical purity of the [11C]9 tracer solution with Sep-Pak purifica-
tion was usually increased higher 10–20% than that without
Sep-Pak purification.18–20
The experimental details and characterization data for com-
pounds 1–10 and for the target tracer [11C]9 are given.26
In summary, an efficient and convenient synthetic route to the
pyrazolopyridine standard 9, normethyl precursor 10 and target
tracer [11C]9 has been developed. An automated self-designed mul-
ti-purpose [11C]-radiosynthesis module for the synthesis of [11C]9
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column chromatography (6:1–4:1 hexanes/EtOAc) to give 4 (18.4 g, 75%) as an
off-white solid, mp 76–77 °C. 1H NMR (CDCl3): d 7.51–7.47 (m, 1H), 7.07–7.03
(m, 1H), 3.97 (s, 3H), 3.67–3.61 (m, 2H), 3.52–3.46 (m, 2H), 1.99–1.91 (m, 4H),
1.10 (t, J = 7.5 Hz, 6H).
(f) 2,6-Difluoro-3-(propylsulfonamido)benzoic acid (5). To
a solution of
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compound (15.0 g, 37.6 mmol) in THF/MeOH (4:1, 150 mL) was added
4
1.0 N NaOH (113 mL, 113.0 mmol). After the reaction mixture was stirred at
room temperature overnight, the majority of the organic solvent was removed
in vacuo. The mixture was cooled with ice bath, and then neutralized with
1.0 N HCl (115 mL) slowly. The precipitate was collected by filtration and
rinsed with water to give 5 (8.42 g, 80%) as a white solid, mp 218–220 °C. 1H
NMR (DMSO-d6): d 14.1 (br s, 1H), 9.75 (s, 1H), 7.56–7.52 (m, 1H), 7.22–7.19
(m, 1H), 3.10–3.08 (m, 2H), 1.78–1.70 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H).
(g) 3-Methoxy-1H-pyrazol-5-amine (6).
A
mixture of 3-amino-5-
hydroxypyrazole (25.0 g, 252.3 mmol) and triphenylphosphine (77.8 g,
296.7 mmol) in CH2Cl2 (400 mL) was cooled to 0 °C and diisopropyl
azodicarboxylate (58.8 mL, 298.6 mmol) was added dropwise at 0 °C to give
a dark brown mobile slurry. The reaction mixture was stirred at 0 °C for 1 h.
Beige slurry formed after 20 min. MeOH (25 mL) was then added dropwise at
0 °C as the slurry thinned considerably to give lighter yellow slurry. After the
reaction mixture was held at 0 °C for 1 h, it was allowed to warm to room
temperature and then stirred at room temperature overnight. Solid was filter
off, the filtrate was concentrated in vacuo. The crude product was purified by
silica gel column chromatography (100:3–100:10 CH2Cl2/MeOH) to give 6
(3.17 g, 11%) as a yellow solid, mp 46–48 °C. 1H NMR (DMSO-d6): d 4.73 (s, 1H),
3.65 (s, 3H). 13C NMR (DMSO-d6): d 162.8, 149.3, 73.0, 55.1. LC/MS (ESI, m/z):
114 ([M+H]+, 100%).
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25. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671.
26. (a) General. All commercial reagents and solvents were purchased from Sigma–
Aldrich and Fisher Scientific, and used without further purification.
(h) 3-Methoxy-5-nitro-1H-pyrazolo[3,4-b]pyridine (7). A mixture of compound 6
(2.0 g, 20.6 mmol) and sodium nitromalonaldehyde monohydrate (3.52 g,
22.4 mmol) in water (70 mL) was heated to 95 °C overnight. The reaction
mixture was cooled to room temperature and acidified with acetic acid to pH 5.
The mixture was extracted with EtOAc, and the combined organic layer was
washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The crude product was purified by silica gel column chromatography
(4:1 hexanes/EtOAc) to give 7 (1.57 g, 39%) as a yellow solid, mp 202–204 °C.
1H NMR (DMSO-d6): d 13.4 (br s, 1H), 9.29 (d, J = 2.5 Hz, 1H), 8.93 (d, J = 2.5 Hz,
1H), 4.07 (s, 3H).
(i) 3-Methoxy-1H-pyrazolo[3,4-b]pyridine-5-amine (8). A solution of compound
7 (1.4 g, 7.2 mmol) in EtOAc/MeOH (1:1, 40 mL) was hydrogenation over 10%
Pd/C (0.8 g) at 60 psi for 7 h. The catalyst was filtered off through a layer of
Celite, and the filtrate was concentrated in vacuo. The crude product was
purified by silica gel column chromatography (100:4 CH2Cl2/MeOH) to give 8
(1.03 g, 87%) as an off-white solid, mp 174–176 °C. 1H NMR (DMSO-d6): d 11.9
(br s, 1H), 8.01 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 2.5, 0.5 Hz, 1H), 4.97 (s, 2H), 3.94
(s, 3H).
[
11C]CH3OTf was prepared according to literature procedure.14 Melting
a
points were determined on a MEL-TEMP II capillary tube apparatus and were
uncorrected. 1H NMR and 13C NMR spectra were recorded at 500 and 125 MHz,
respectively, on
a Bruker Avance II 500 MHz NMR spectrometer using
tetramethylsilane (TMS) as an internal standard. Chemical shift data for the
proton resonances were reported in parts per million (ppm, d scale) relative to
internal standard TMS (d 0.0), and coupling constants (J) were reported in hertz
(Hz). LC–MS analysis was performed on an Agilent system, consisting of an
1100 series HPLC connected to a diode array detector and a 1946D mass
spectrometer configured for positive-ion/negative-ion electrospray ionization.
The high resolution mass spectra (HRMS) were obtained using a Waters/
Micromass LCT Classic spectrometer. Chromatographic solvent proportions are
indicated as volume: volume ratio. Thin-layer chromatography (TLC) was run
using Analtech silica gel GF uniplates (5 ꢀ 10 cm2). Plates were visualized
under UV light. Normal phase flash column chromatography was carried out
on EM Science silica gel 60 (230–400 mesh) with a forced flow of the indicated
solvent system in the proportions described below. All moisture- and air-
sensitive reactions were performed under a positive pressure of nitrogen
maintained by a direct line from a nitrogen source. Analytical HPLC was
(j) 2,6-Difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfo-
namido)benzamide (9).
A mixture of compound 5 (682 mg, 2.44 mmol),
compound
8
(400 mg, 2.44 mmol), HOBtꢁH2O (374 mg, 2.44 mmol), EDCI
(443 mg, 2.44 mmol) in DMF (10 mL) was stirred at room temperature
overnight. The solvent was removed in vacuo to give a dark viscous mixture.
A solution of water/saturated aqueous NaHCO3 (10 mL) was added dropwise
with rapid stirring at 0 °C. The mixture was stirred at 0 °C for 1 h, and the
precipitate was collected by filtration and rinsed with water to give a tan solid.
The crude product was purified by silica gel column chromatography (100:4–
100:10 CH2Cl2/MeOH) to give 9 (861 mg, 83%) as a beige solid, mp 206–207 °C.
1H NMR (DMSO-d6): d 12.6 (br s, 1H), 11.1 (s, 1H), 9.81(s, 1H), 8.59 (d,
J = 2.0 Hz, 1H), 8.49 (d, J = 2.0 Hz, 1H), 7.59–7.54 (m, 1H), 7.28 (t, J = 8.5 Hz, 1H),
4.01 (s, 3H), 3.14–3.11 (m, 2H), 1.81–1.73 (m, 2H), 0.99 (t, J = 7.5 Hz, 3H). 13C
NMR (DMSO-d6): d 158.0, 154.7, 149.7, 143.4, 128.0, 118.4, 102.4, 55.7, 53.8,
16.9, 12.6. LC/MS (ESI, m/z): 426 ([M+H]+, 100%). HRMS (ESI, m/z): calcd for
performed using a Prodigy (Phenomenex) 5
mobile phase 1:1 CH3CN/H2O; flow rate 1.5 mL/min; and UV (254 nm) and
-ray (PIN diode) flow detectors. Semi-preparative HPLC was performed using
a Prodigy (Phenomenex) 5
m C-18 column, 12 nm, 10 ꢀ 250 mm; mobile
phase 1:1 CH3CN/H2O; flow rate 5.0 mL/min; UV (254 nm) and -ray (PIN
diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from Waters
lm C-18 column, 4.6 ꢀ 250 mm;
c
l
c
Corporation (Milford, MA). Sterile Millex-FG 0.2
from Millipore Corporation (Bedford, MA).
lm filter units were obtained
(b) Methyl 2,6-difluorobenzoate (1). To a solution of 2,6-difluorobenzoic acid
(30.0 g, 190.0 mmol) in MeOH (100 mL) was added concentrated sulfuric acid
(5 mL) dropwise at room temperature. The reaction mixture was heated under
reflux overnight. The solvent was removed in vacuo. The residue was dissolved
in EtOAc and washed with saturated NaHCO3 and brine. The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give 1
(23.2 g, 71%) as a yellow oil. 1H NMR (CDCl3): d 7.45–7.39 (m, 1H), 6.98–6.93
(m, 2H), 3.96 (s, 3H).
C
17H17N5O4SF2Na ([M+Na]+) 448.0867; found 448.0849.
(k) 2,6-Difluoro-N-(3-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfo-
namido)benzamide (10). To a suspension of compound 9 (200 mg, 0.47 mmol)
and NaI (290 mg, 1.93 mmol) in acetonitrile (4 mL) was added
chlorotrimethylsilane (0.24 mL, 1.93 mmol) dropwise under nitrogen
atmosphere. The reaction mixture was heated under reflux overnight. The
reaction was quenched with MeOH (2 mL). The solvent was removed in vacuo,
and the residue was dissolved in MeOH and purified via reversed-phase semi-
preparative HPLC using a Shimadzu LC-20AT pump, a SPD-M20A diode array
(c) Methyl 2,6-difluoro-3-nitrobenzoate (2). To a solution of compound 1 (21.0 g,
122.0 mmol) in concentrated sulfuric acid (50 mL) was added fuming nitric
acid (8 mL) dropwise at 0 °C. After the reaction mixture was stirred at 0 °C for
1 h, it was poured into ice-water. The precipitate was collected by filtration
detector (DAD), a Luna C18 column (10 ꢀ 250 mm, 5
lm), a gradient mobile
and rinsed with water to give 2 (26.0 g, 98%) as a white solid, mp 58–60 °C. 1
NMR (CDCl3): d 8.25–8.22 (m, 1H), 7.15–7.11 (m, 1H), 4.01 (s, 3H).
H
phase composed of 20% CH3CN (0.1% TFA)—80% H2O (0.1% TFA) to 60% CH3CN
(0.1% TFA)—40% H2O (0.1% TFA), flow rate 5.0 mL/min and UV 254 nm. The
product fraction was collected at ꢂ10 min to afford 10 (135 mg, 70%) as a pale
pink solid, mp 265–267 °C. 1H NMR (DMSO-d6): d 12.6 (br s, 1H), 11.0 (s, 1H),
9.81(s, 1H), 8.58 (d, J = 1.5 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H), 7.59–7.54 (m, 1H),
7.28 (t, J = 8.5 Hz, 1H), 3.14 (t, J = 7.5 Hz, 1H), 1.82–1.74 (m, 2H), 1.00 (t,
J = 7.5 Hz, 3H). 13C NMR (CD3OD): d 160.8, 158.3, 152.6, 146.2, 129.5, 123.7,
107.0, 55.2, 18.3, 13.1. LC/MS (ESI, m/z): 412 ([M+H]+, 100%). HRMS (ESI, m/z):
calcd for C16H15N5O4SF2Na ([M+Na]+) 434.0711; found 434.0694.
(d) Methyl 3-amino-2,6-difluorobenzoate (3). A solution of compound 2 (15.0 g,
69.1 mmol) in EtOH (250 mL) was hydrogenation over 10% Pd/C (4.0 g) at
50 psi for 22 h. The catalyst was filtered off through a layer of Celite, and the
filtrate was concentrated in vacuo. The crude product was purified by silica gel
column chromatography (4:1–3:1 hexanes/EtOAc) to give 3 (12.9 g, 93%) as a
pale yellow oil. 1H NMR (DMSO-d6): d 6.93–6.91 (m, 2H), 5.26 (br s, 2H), 3.87
(s, 3H).
(e) Methyl 2,6-difluoro-3-(N-(propylsulfonyl)propylsulfonamido)benzoate (4). To
a solution of compound 3 (11.5 g, 61.4 mmol) and triethylamine (25.67 mL,
184.2 mmol) in CH2Cl2 (55 mL) was added propane-1-sulfonyl chloride
(17.25 mL, 153.3 mmol) dropwise at 0 °C. The reaction mixture was stirred at
room temperature for 1 h. Water (150 mL) was added, and the organic layer
was separated, washed with water, brine, dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The crude product was purified by silica gel
(l) 2,6-Difluoro-N-(3-[11C]methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propyl-
sulfonamidio)benzamide ([11C]9). [11C]CO2 was produced by the 14N(p, 11C
a)
nuclear reaction in the small volume (9.5 cm3) aluminum gas target provided
with the Siemens RDS-111 Eclipse cyclotron. The target gas consisted of 1%
oxygen in nitrogen purchased as a specialty gas from Praxair, Indianapolis, IN.
Typical irradiations used for the development were 55
lA beam current and
15 min on target. The production run produced approximately 28.5 GBq of

M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1017–1021
1021
[
11C]CO2 at EOB. In a small reaction vial (5 mL), the precursor 10 (0.3–0.5 mg)
was dissolved in CH3CN (300 L). To this solution was added 2 N NaOH (3 L).
No carrier-added (high specific activity) [11C]CH3OTf that was produced by the
gas-phase production method14 from 11C]CO2 through 11C]CH4 and
11C]CH3Br with silver triflate (AgOTf) column was passed into the reaction
30 mL water. The diluted tracer solution was then passed through a C-18 Sep-
Pak Plus cartridge, and washed with water (5 mL ꢀ 4). The cartridge was eluted
with EtOH (1 mL ꢀ 2), followed by 10 mL saline, to release [11C]9. The eluted
l
l
[
[
product was then sterile-filtered through a Millex-FG 0.2 lm membrane into a
[
sterile vial. Total radioactivity was assayed and total volume was noted for
tracer dose dispensing. Retention times in the semi-preparative HPLC system
vial at room temperature, until radioactivity reached a maximum (ꢂ2 min),
and then the reaction vial was isolated and heated at 80 °C for 3 min. The
contents of the reaction vial were diluted with NaHCO3 (0.1 M, 1 mL), and
injected onto the semi-preparative HPLC column with 3 mL injection loop for
purification. The product fraction was collected in a recovery vial containing
were: tR 10 = 5.87 min, tR 9 = 8.32 min, tR [
11C]9 = 8.32 min. Retention times in
the analytical HPLC system were: tR 10 = 2.28 min, tR 9 = 4.17 min, tR
[
[
11C]9 = 4.17 min. The decay corrected radiochemical yield of 11C]9 from
[
11C]CO2 was 40–50%.