Synthesis of (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]methoxy- 7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one as a new potential PET probe for imaging of the enzyme PIM1

Article abstract of DOI:10.1016/j.bmcl.2013.05.091

(Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl) benzofuran-3(2H)-one is a potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor with an IC₅₀ value of 3 nM. (Z)-2-((1H-Indazol-3-yl)methylene)-6-[¹¹C]methoxy-7- (piperazin-1-ylmethyl)benzofuran-3(2H)-one, a new potential PET probe for imaging of the enzyme PIM1, was first designed and synthesized in 20-30% decay corrected radiochemical yield and 370-740 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled Boc-protected intermediate followed by a quick acidic de-protection.

Full text of DOI:10.1016/j.bmcl.2013.05.091

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4342–4346
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]
methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one
as a new potential PET probe for imaging of the enzyme PIM1
Mingzhang Gao a, Min Wang 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:
(Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one is
a
Received 9 May 2013
Revised 22 May 2013
Accepted 28 May 2013
Available online 6 June 2013
potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor
with an IC50 value of 3 nM. (Z)-2-((1H-Indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylmethyl)
benzofuran-3(2H)-one, a new potential PET probe for imaging of the enzyme PIM1, was first designed and
synthesized in 20–30% decay corrected radiochemical yield and 370–740 GBq/lmol specific activity at end
of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled Boc-protected interme-
diate followed by a quick acidic de-protection.
Keywords:
(Z)-2-((1H-Indazol-3-yl)methylene)-6-
Ó 2013 Elsevier Ltd. All rights reserved.
[
11C]methoxy-7-(piperazin-1-
ylmethyl)benzofuran-3(2H)-one ([11C]4)
Proviral integration site in moloney murine
leukemia virus kinase 1 (PIM1)
Radiosynthesis
Positron emission tomography (PET)
Alzheimer’s disease
Cancer
Protein kinase (PK) is a family of enzymes that are involved in
controlling the function of other proteins through the phosphoryla-
tion of hydroxyl groups of serine and threonine and/or tyrosine
amino acid residues on these proteins.1 Most PKs act on both serine
and threonine (serine/threonine kinases), others act on tyrosine
(tyrosine kinases), and a number act on all three (dual-specificity
kinases), and these PKs regulate all the critical phases of cell growth
including cell proliferation, differentiation, and apoptosis.1 Proviral
integration site in moloney murine leukemia virus kinase 1 (PIM1)
is a member of serine/threonine kinase.2 PK expression and activity
are associated with various disease processes, such as Alzheimer’s
disease and cancer.3 In cancer, PIM1 phosphorylate multiple pro-
teins as well as drug efflux transporters, resulting in drug resis-
tance.2 Overexpression of PIM1 in cancers indicates PIM1 is an
emerging target for cancer therapy.4,5 PIM1 is an attractive target
for cancer imaging as well, however, so far no specific cancer imag-
ing agent is developed for PIM1.6 We are interested in development
of new cancer imaging agents for the biomedical imaging technique
positron emission tomography (PET) to image cancer and to moni-
tor the response of cancer to therapy. In our previous work, we have
developed a series of radiolabeled (carbon-11 and fluorine-18) PK
inhibitors such as [11C]MKC-1 ([11C]Ro 31-7453), [11C]SB-216763,
[
11C]Enzastaurin ([11C]LY317615), [11C]GSK2126458, 2-[18F]GSK
2126458, 4-[18F]GSK2126458, [11C]Gefinitib ([11C]Iressa), N-[11C]
Vandetanib and O-[11C]Vandetanib, as shown in Figure 1, as new
cancer imaging agents for PET to study PK and PK inhibitors.7–12
In this ongoing study, we target PIM1. (Z)-2-((1H-Indazol-3-
yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)benzofuran-
3(2H)-one (4) is a potent and selective PIM1 inhibitor with an IC50
value of 3 nM, recently developed by Nakano et al.2 To radiolabel
therapeutic agents as diagnostic agents for imaging of PK and mon-
itoring of therapeutic efficacy of PK inhibitors, we have designed
and synthesized (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]meth-
oxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one ([11C]4) as a
new potential PET probe, for the first time.
The target compound 4, and its Boc-protected precursor 5 and
intermediate 3 were synthesized by adopting the available litera-
ture methods from previous work.2 As illustrated in Scheme 1,
Mannich reaction of 6-hydroxybenzofuran-3(2H)-one and tert-
butyl piperazine-1-carboxylate with paraformaldehyde in EtOH
at reflux provided compound 1 in 62% yield. The yield was
improved by eliminating microwave irradiation. Mitsunobu cou-
pling reaction of 1 and MeOH with diisopropyl azodicarboxylate
⇑
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.091

M. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4342–4346
4343
H
N
H
N
O
O
O
O
O2N
N
N
11CH3
O2N
N
11CH3
N
CH3
CH3
[
11C]MKC-1 ([11C]Ro 31-7453)
H
N
O
O
H
N
N
N
N
O
O
11CH3
Cl
Cl
N
11CH3
N
11C]SB-216763
[
11C]Enzastaurin ([11C]LY317615)
[
N
N
N
N
N
N
N
H311CO
N
H3CO
N
H3CO
O
N
O
O
O
O
H311C
O
O
S
S
S
N
H
F
N
N
H
H
18F
F
F
18F
N
F
N
11C]GSK2126458
4-[18F]GSK2126458
2-[18F]GSK2126458
[
F
Br
F
Br
F
HN
HN
O
HN
Cl
O
O
O
N
O
N
N
N
H311C
O
N
N
O
N
N
N
H311C
N-[11C]Vandetanib
O-[11C]Vandetanib
[
11C]Gefinitib ([11C]Iressa)
H3C
Figure 1. Radiolabeled PK inhibitors.
O
O
N
O
O
N
O
HO
O
N
N
N
i
ii
+
HO
O
O
O
N
O
H
O
O
1, 62%
2, 42% or 81%
O
HN
O
N
N
NH
N
N
iii
iv
NH
N
O
O
O
O
O
O
4, 83%
3, 70%
Scheme 1. Synthesis of reference standard 4 and Boc-protected intermediate 3. Reagents and conditions: (i) paraformaldehyde, EtOH, reflux; (ii) MeOH, DIAD, PPh3, THF,
room temperature (RT); or CH2N2, THF, RT; (iii) 1H-indazole-3-carbaldehyde, piperidine, MeOH, reflux; (iv) TFA, CH2Cl2, RT.
(DIAD) and triphenylphosphine as catalysts afforded 2 in 42% yield.
This methylation method gave low yield, because it generated a
few side products and it was difficult to work up. Alternative meth-
ylation method of 1 with diazomethane13 was adopted to improve
the yield, which afforded 2 in 81% yield. Another methylation
method using typical methylating agent CH3I was tested as well.
However, it gave poor chemical yield. Knoevenagel condensation
of 2 with 1H-indazole-3-carbaldehyde provided 3 in 70% yield.
Subsequent acidic removal of Boc group using trifluoroacetic acid
(TFA) obtained reference standard 4 in 83% yield.
As outlined in Scheme 2, a new synthetic route was designed to
synthesize new compound Boc-protected precursor 5 and interme-
diate 3. First Knoevenagel condensation of 1 with 1H-indazole-3-
carbaldehyde gave precursor 5 in 71% yield, then methylation of
5 in two different ways aforementioned furnished 3 in 41% and
80% yield, respectively.

4344
M. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4342–4346
O
O
O
O
O
N
N
O
N
N
N
i
ii
N
NH
N
NH
HO
O
N
O
O
HO
O
O
O
O
5, 71%
1
3, 41% or 80%
Scheme 2. Synthesis of Boc-protected precursor 5 and Boc-protected intermediate 3. Reagents and conditions: (i) 1H-indazole-3-carbaldehyde, piperidine, MeOH, reflux; (ii)
MeOH, DIAD, PPh3, THF, RT; or CH2N2, THF, RT.
Synthesis of the target radiotracer [11C]4 is indicated in Scheme
3. Boc-protected precursor 5 was labeled by [11C]methyl triflate
([11C]CH3OTf) prepared from [11C]CO2,14,15 in the presence of 2 N
NaOH in acetonitrile through the O-[11C]methylation10–12 to pro-
vide a radiolabeling Boc-protected intermediate [11C]3. Without
further purification, [11C]3 was quickly converted to the target tra-
cer [11C]4 by acidic removal of Boc group under 1 N HCl. [11C]4 was
isolated by a semi-preparative high performance liquid chroma-
tography (HPLC) with a Prodigy C-18 column from Phenomenex
and a solid-phase extraction (SPE) with a disposable C-18 Plus
Sep-Pak cartridge from Waters (a second purification or isolation
process)10,16,17 to produce the pure radiolabeled compound in
20–30% radiochemical yield, decay corrected to end of bombard-
ment (EOB), based on [11C]CO2. The radiolabeling yield was rela-
tively low, since it was a one-pot two-step radiolabeling reaction,
but it was similar to the values previously reported by our lab
for the tracers synthesized from the same synthetic strategy.7–9
Addition of NaHCO3 to quench the radiolabeling reaction and to di-
lute the radiolabeling mixture prior to the injection onto the semi-
preparative HPLC column for purification gave better separation of
was 30–40 min from EOB. The SA was in a range of 370–
740 GBq/ mol at EOB. The SA can also be measured by analytical
l
HPLC, which is consistent with the on-the-fly technique to deter-
mine the SA by semi-preparative HPLC. Chemical purity and radio-
chemical purity were determined by analytical HPLC.22 The
chemical purity of the precursor 5, intermediate 3 and reference
standard 4 was >95%. The radiochemical purity of the target tracer
[
11C]4 was >99% determined by radio-HPLC through
c
-ray (PIN
diode) flow detector, and the chemical purity of the target tracers
11C]4 was >90% determined by reversed-phase HPLC through UV
[
flow detector.
SA is defined as the radioactivity per unit mass of a radionuclide
or a labeled compound. SA (MBq/mg) = 3.13 Â 109/A Â t1/2, where A
is the mass number of the radionuclide, and t1/2 is the half-life in
hours of the radionuclide. For carbon-11, carrier-free 11C, maximum
(theoretical) 11C SA = 340,918 GBq/ mol.23 Actual SAs of the
l
11C-tracers in the PET chemistry facility are depended on two parts:
(1) carrier from the 11C-target, and (2) carrier from the 11C-radio-
synthesis unit.24,25 Furthermore, actual SA for the 11C-tracers
synthesized by 11C-methylation with [11C]CH3OTf in our PET chem-
istry facility is depended on two parts: (1) carrier from the cyclotron
consisted of the 11C gas irradiation target system, and (2) carrier
from the [11C]methyl triflate ([11C]CH3OTf) system, 11C radiolabeled
precursor or called 11C radiolabeled methylating reagent. The 11C
gas target we used is the Siemens RDS-111 Eclipse cyclotron 11C
gas target. The [11C]CH3OTf production system we used is an Eckert
& Ziegler Modular Lab C-11 Methyl Iodide/Triflate module, conve-
nient gas phase bromination of [11C]methane and production of
[
11C]4 from its radiolabeled intermediate [11C]3 and normethyl
precursor 5.10,16,17 The radiosynthesis was performed in a home-
built automated multi-purpose 11C-radiosynthesis module, allow-
ing measurement of specific radioactivity during synthesis.18–20
This 11C-radiosynthesis module includes the overall design of the
reaction, purification and reformulation capabilities of the proto-
type system. In addition, the specific activity (SA, GBq/lmol at
EOB) of 11C-tracer can be automatically determined prior to prod-
uct delivery for compounds purified by the HPLC-portion of the
system.21 Briefly, analysis of the chromatographic data utilized
PeakSimple software (SRI Instruments, Las Vegas, NV, USA). Imme-
diately 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 calibration curve previously constructed using the same
detector, loop and flow rate. The mass peak from the UV chromato-
gram (without decay correction) is similarly compared to a stan-
dard 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 mass peak (in nmoles) gives SA at EOB in GBq/
[
11C]CH3OTf, a ‘dry’ method using Br2 different with other ‘dry’
method using I2 and ‘wet’ method using LiAlH4 and HI. Our system
will have much less 12C carrier-added in comparison with other ‘dry’
method and ‘wet’ method.15 The SA for our 11C-tracers usually
ranges from 370 to 925 GBq/
works. Our study has proved that the maximum in-target 11C SA is
mol (25 Ci/ mol) at EOB in our cyclotron and
11C]CH3OTf system. The SA of the title tracer was 370–740 GBq/
lmol at EOB according to our previous
ꢀ925 GBq/
l
l
[
lmol at EOB, which was similar to the values previously reported
by our lab. A historical perspective on the SA of radiopharmaceuti-
cals indicated it is difficult to achieve high SA for the 11C-tracers.23
However, a recent paper reported the maximum in-target 11C SA
lmol. The overall synthesis, purification and reformulation time
2775 GBq/
l
mol (75 Ci/
l
mol) at EOB has been obtained, using GE
O
O
O
N
HN
H311CO
O
N
N
N
i
ii
NH
N
N
NH
N
H311CO
NH
N
O
O
HO
O
O
O
[
11C]3
O
[
11C]4, 20-30%
5
Scheme 3. Synthesis of target tracer [11C]4. Reagents and conditions: (i) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 °C, 3 min; (ii) 1 N HCl, 80 °C, 3 min.

M. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4342–4346
4345
26. Nabulsi, N. B.; Zheng, M.-Q.; Ropchan, J.; Labaree, D.; Ding, Y.-S.; Blumberg, L.;
Huang, Y. Nucl. Med. Biol. 2011, 38, 215.
PETtrace cyclotron and TRACERLab FXC automated synthesizer for
the synthesis of a 11C-tracer, [11C]GR103545.26
27. (a) General. All commercial reagents and solvents were purchased from
Sigma–Aldrich and Fisher Scientific, and used without further purification.
The experimental details and characterization data for com-
pounds 1–5 and for the tracer [11C]4 are given.27
[
11C]CH3OTf was prepared according to a literature procedure.15 Melting points
were determined on
a MEL-TEMP II capillary tube apparatus and were
In summary, a carbon-11-labeled PIM1 inhibitor (Z)-2-((1H-
indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylme-
thyl)benzofuran-3(2H)-one ([11C]4) was first designed and synthe-
sized as a new potential PET probe for imaging of the enzyme
PIM1. An automated multi-purpose 11C-radiosynthesis module of
our own design for fully automated synthesis of [11C]4 has been
built, featuring the measurement of specific activity by the on-
the-fly technique. The radiosynthesis employed a one-pot two-step
reaction via O-[11C]methylation radiolabeling on hydroxyl group of
the Boc-protected phenolic precursor followed by a quick acidic
removal of Boc group, incorporated efficiently with the most com-
monly used [11C]methylating agent, [11C]CH3OTf, produced by
gas-phase production of [11C]methyl bromide ([11C]CH3Br) from
our laboratory. The target tracer was isolated and purified by a
semi-preparative HPLC procedure in moderate radiochemical
yields, short overall synthesis time, and high specific activity.
These results facilitate the potential preclinical and clinical PET
studies of radiolabeled PIM1 inhibitor [11C]4 in animals and
humans.
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 Â 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
performed using a Prodigy (Phenomenex) 5
mobile phase 3:1:3 CH3CN/MeOH 20 mM phosphate buffer solution (pH 6.7);
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 Â 250 mm; mobile phase 40% CH3CN/60% 0.1 M NH4OAc
(pH 6.7); flow rate 5.0 mL/min; UV (254 nm) and -ray (PIN diode) flow
detectors. C18 Plus Sep-Pak cartridges were obtained from Waters Corporation
lm C-18 column, 4.6 Â 250 mm;
c
l
c
(Milford, MA). Sterile Millex-FG 0.2
Millipore Corporation (Bedford, MA).
lm filter units were obtained from
Acknowledgments
(b)
tert-Butyl
4-((6-hydroxy-3-oxo-2,3-dihydrobenzofuran-7-yl)methyl)
piperazine-1-carboxylate (1). A mixture of 6-hydroxybenzofuran-3(2H)-one
(14.82 g, 98.7 mmol), tert-butyl piperazine-1-carboxylate (18.39 g, 98.7 mmol)
and paraformaldehyde (3.05 g, 101.66 mmol) in EtOH (150 mL) was heated to
reflux for 5 h. The mixture was evaporated under reduced pressure, and the
resulting residue was purified by column chromatography (10–100% EtOAc/
hexanes) on silica gel to afford 1 (21.29 g, 62%) as a white solid; Rf = 0.70 (5%
MeOH/CH2Cl2); mp 123–125 °C. 1H NMR (DMSO-d6): d 1.38 (s, 9H, 3 Â CH3),
2.44 (t, J = 5.0 Hz, 4H, 2 Â CH2), 3.32 (br s, 4H, 2 Â CH2), 3.67 (s, 2H, CH2), 4.73
(s, 2H, CH2), 6.58 (d, J = 8.5 Hz, 1H, Ph–H), 7.39 (d, J = 8.5 Hz, Ph–H). MS (ESI):
349 ([M+H]+, 100%).
This work was partially supported by the Breast Cancer Re-
search Foundation and Indiana State Department of Health (ISDH)
Indiana Spinal Cord & Brain Injury Fund(ISDH EDS-A70-2-079612).
1H and 13C NMR spectra were recorded on a Bruker Avance II
500 MHz NMR spectrometer in the Department of Chemistry and
Chemical Biology at Indiana University Purdue University India-
napolis (IUPUI), which is supported by a NSF-MRI grant CHE-
0619254.
(c)
tert-Butyl
4-((6-methoxy-3-oxo-2,3-dihydrobenzofuran-7-yl)methyl)
piperazine-1-carboxylate (2). Method A. Diisopropyl azodicarboxylate
(3.03 g, 15.0 mmol) in THF (5.0 mL) was slowly added into a mixture of
compound
1
(3.48 g,
10.0 mmol),
MeOH
(598 mg,
13.0 mmol),
References and notes
triphenylphosphine (3.93 g, 15.0 mmol) in THF (80 mL) at 0 °C. Then the
reaction mixture was stirred at room temperature (RT) for 2 h, and
concentrated in vacuo. The resulting residue was purified by column
chromatography (0–3% MeOH/CH2Cl2) on silica gel to obtain 2 (1.52 g, 42%)
as a white solid; Rf = 0.42 (5% MeOH/CH2Cl2); mp 68–70 °C. 1H NMR (CDCl3): d
1.44 (s, 9H, 3 Â CH3), 2.48 (br s, 4H, 2 Â CH2), 3.42–3.43 (m, 4H, 2 Â CH2), 3.71
(s, 2H, CH2), 3.93 (s, 3H, OCH3), 4.64 (s, 2H, CH2), 6.69 (d, J = 8.5 Hz, 1H, Ph–H),
7.61 (d, J = 8.5 Hz, 1H, Ph–H). MS (ESI): 363 ([M+H]+, 100%). Method B. To a
solution of compound 1 (696 mg, 2.0 mmol) in THF (50 mL), diazomethane
(18 mmol) in ether was added at 0 °C, and the reaction mixture was stirred at
RT for 1 h, and concentrated in vacuo. The resulting residue was purified
through column chromatography (0–3% MeOH/CH2Cl2) on silica gel to afford 2
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(586 mg, 81%) as
aforementioned.
a white solid. The analytical data were same as
(d) (Z)-tert-Butyl 4-((2-((1H-indazol-3-yl)methylene)-6-methoxy-3-oxo-2,3-
dihydrobenzofuran-7-yl)methyl)piperazine-1-carboxylate (3). Method for
C
preparation of 3 from 2. A mixture of compound 2 (1.81 g, 5.0 mmol), 1H-
indazole-3-carbaldehyde (731 mg, 5.0 mmol), and piperidine (426 mg,
5.0 mmol) in MeOH (40 mL) was stirred at 60 °C for 3 h. Then the reaction
was evaporated in reduced pressure, and the resulting residue was purified by
column chromatography (0–8% MeOH/CH2Cl2) on silica gel to afford 3 (1.72 g,
70%) as a yellow solid; Rf = 0.40 (5% MeOH/CH2Cl2); mp 228–231 °C. 1H NMR
(DMSO-d6): d 1.37 (s, 9H. 3 Â CH3) 2.46 (t, J = 4.5 Hz, 4H, 2 Â CH2), 3.29 (br s,
4H, 2 Â CH2), 3.74 (s, 2H, CH2), 3.97 (s, 3H, OCH3), 7.05 (d, J = 8.5 Hz, 1H, Ph–H),
7.08 (s, 1H, CH@), 7.26 (t, J = 8.0 Hz, 1H, Ph–H), 7.47 (t, J = 8.0 Hz, 1H, Ph–H),
7.64 (d, J = 8.0 Hz, 1H, Ph–H), 7.79 (d, J = 8.5 Hz, 1H, Ph–H), 8.59 (d, J = 8.0 Hz,
1H, Ph–H), 13.86 (s, 1H, NH); 1H NMR (CDCl3): d 1.42 (s, 9H, 3 Â CH3), 2.59 (br
s, 4H, 2 Â CH2), 3.45 (br s, 4H, 2 Â CH2), 3.88 (s, 2H, CH2), 3.97 (s, 3H, OCH3),
6.81 (d, J = 8.5 Hz, 1H, Ph–H), 7.24–7.27 (m, 2H, Ph–H and CH@), 7.41 (dt,
J = 0.5, 7.0 Hz, 1H, Ph–H), 7.52 (d, J = 8.0 Hz, 1H, Ph–H), 7.79 (d, J = 8.5 Hz, 1H,
Ph–H), 8.39 (d, J = 7.5 Hz, 1H, Ph–H), 11.50 (br s, 1H, NH). MS (ESI): 491
([M+H]+, 100%); MS (ESI), 489 ([MÀH]À, 100%). Method D for preparation of 3
from 5. The title compound was obtained in 41% and 80% yield, respectively, in
the same manner as described for 2. The analytical data were same as
aforementioned.
19. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd.
Radiopharm. 2005, 48, S224.
20. Wang, M.; Gao, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 965.
21. Gao, M.; Shi, Z.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 1953, 23.
22. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671.
23. Lapi, S. E.; Welch, M. J. Nucl. Med. Biol. 2012, 39, 601.
24. Gao, M.; Wang, M.; Miller, K. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 1558.
25. Gao, M.; Yang, Q.; Wang, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Appl.
Radiat. Isot. 2013, 74, 61.
(e) (Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)
benzofuran-3(2H)-one (4). A mixture of compound 3 (0.49 g, 1.0 mmol) in
the solution of TFA (2 mL) and CH2Cl2 (8 mL) was stirred at RT for 3 h. Then the
mixture was evaporated under reduced pressure. The residue was added

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M. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4342–4346
aqueous NaHCO3 to adjust pH to 8, and the mixture was extracted with CH2Cl2
time was 30 min. The production run produced approximately 45.5 GBq of
11C]CO2 at EOB. The precursor
(0.1–0.3 mg) was dissolved in CH3CN
(300 L). To this solution was added 2 N NaOH (3 L). The mixture was
(50 mL Â 3), dried over Na2SO4, and concentrated. The crude was purified by
[
5
column chromatography (5–20% MeOH/CH2Cl2) on silica gel to afford 4 (0.32 g,
83%) as a yellow solid; Rf = 0.12 (10% MeOH/CH2Cl2); mp >300 °C. 1H NMR
(DMSO-d6): d 2.50 (m, 4H, 2 Â CH2), 2.81 (br s, 4H, 2 Â CH2), 3.92 (s, 2H, CH2),
3.99 (s, 3H, OCH3), 7.10 (d, J = 8.5 Hz, 1H, Ph–H), 7.13 (s, 1H, CH@), 7.30 (t,
J = 7.5 Hz, 1H, Ph–H), 7.47 (t, J = 7.5 Hz, 1H, Ph–H), 7.64 (d, J = 8.0 Hz, 1H, Ph–
H), 7.84 (d, J = 8.5 Hz, 1H, Ph–H), 8.52 (d, J = 8.0 Hz, 1H, Ph–H), 13.87 (br s, 1H,
NH). MS (ESI): 391 ([M+H]+, 100%); MS (ESI): 389 ([MÀH]À, 100%).
l
l
transferred to a small reaction vial. No-carrier-added (high specific activity)
[
[
11C]CH3OTf that was produced by the gas-phase production method15 from
11C]CO2 through [11C]CH4 and [11C]CH3Br with AgOTf column was passed into
the reaction vial at RT until radioactivity reached a maximum (ꢀ2 min), and
then the reaction vial was isolated and heated at 80 °C for 3 min to produce (Z)-
tert-butyl
dihydrobenzofuran-7-yl)methyl)piperazine-1-carboxylate ([11C]3). Then,
solution of 1 N HCl (500 L) was introduced to the reaction vial. The reaction
4-((2-((1H-indazol-3-yl)methylene)-6-[11C]methoxy-3-oxo-2,3-
(f) (Z)-tert-Butyl 4-((2-((1H-indazol-3-yl)methylene)-6-hydroxy-3-oxo-2,3-
dihydrobenzofuran-7-yl)methyl)piperazine-1-carboxylate (5). The title
compound was obtained in the same manner as described for 3 (Method C).
From compound 1 (3.48 g, 10.0 mmol), 1H-indazole-3-carbaldehyde (1.46 g,
10.0 mmol), and piperidine (852 mg, 10.0 mmol), 5 (3.38 g, 71%) was obtained
as a yellow solid; Rf = 0.43 (5% MeOH/CH2Cl2); mp >300 °C. 1H NMR (DMSO-
d6): d 1.37 (s, 9H, 3 Â CH3), 2.50–2.53 (m, 4H, 2 Â CH2), 3.34 (br s, 4H, 2 Â CH2),
3.82 (s, 2H, CH2), 6.78 (d, J = 8.0 Hz, 1H, Ph–H), 7.05 (s, 1H, CH@), 7.27 (t,
J = 7.5 Hz, 1H, Ph–H), 7.47 (t, J = 7.5 Hz 1H, Ph–H), 7.60 (d, J = 8.0 Hz, 1H, Ph–H),
7.63 (d, J = 8.0 Hz, 1H, Ph–H), 8.54 (d, J = 8.0 Hz, 1H, Ph–H), 13.82 (s, 1H, NH).
13C NMR (DMSO-d6): d 5.99, 27.93, 50.09, 52.28, 78.74, 99.42, 106.83, 112.72,
121.42, 121.52, 121.64, 124.63, 126.66, 146.10, 153.62, 165.44, 166.34, 181.34.
MS (ESI): 477 ([M+H]+, 100%); MS (ESI): 475 ([MÀH]À, 100%). HRMS (ESI): calcd
for C26H29N4O5, 477.2138 ([M+H]+); found 477.2134.
a
l
mixture was sealed 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 30 mL water. The diluted
tracer solution was then passed through a C-18 Sep-Pak Plus cartridge, and
washed with water (5 mL Â 4). The cartridge was eluted with EtOH (1 mL Â 2),
followed by 10 mL saline, to release [11C]4. The eluted product was then
sterile-filtered through a sterile vented Millex-GS 0.22 lm cellulose acetate
membrane, and collected into a sterile vial. Total radioactivity (2.3–4.9 GBq)
was assayed and total volume (10–11 mL) was noted for tracer dose
dispensing. The overall synthesis, purification and formulation time was 30–
40 min from EOB. Retention times in the analytical HPLC system were: tR
(g)
ylmethyl)benzofuran-3(2H)-one ([11C]4).
14N(p,)11 nuclear reaction in the small volume (9.5 cm3) aluminum gas
(Z)-2-((1H-Indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-
5 = 4.27 min, tR 3 = 5.16 min, tR 4 = 3.23 min, tR
11C]4 = 3.23 min. Retention times in the semi-preparative HPLC system
were: tR 5 = 4.12 min, tR 3 = 9.35 min, tR 4 = 6.78 min, tR
11C]3 = 9.35 min,
and tR
11C]4 = 6.78 min. The radiochemical yields were 20–30% decay
corrected to EOB, based on [11C]CO2.
[
11C]3 = 5.16 min, and tR
[
11C]CO2 was produced by the
[
C
[
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, USA. The proton-beam current was 55 lA, and the irradiation