Synthesis of [11C]CX-6258 as a new PET tracer for imaging of Pim kinases in cancer

Article abstract of DOI:10.1016/j.bmcl.2015.07.061

Abstract The reference standard CX-6258 {(E)-5-chloro-3-((5-(3-(4-methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)indolin-2-one, 4a} and its desmethylated precursor N-desmethyl-CX-6258 {(E)-3-((5-(3-(1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)-5-chloroindolin-2-one, 5} for radiolabeling were synthesized from 5-bromo-2-furaldehyde and 3-carboxybenzeneboronic acid in 3 and 4 steps with 29-49% and 24-32% overall chemical yield, respectively. The target tracer [¹¹C]CX-6258 {(E)-5-chloro-3-((5-(3-(4-[¹¹C]methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)indolin-2-one, [¹¹C]4a} was prepared from N-desmethyl-CX-6258 (5) with [¹¹C]CH₃OTf under basic condition (2 N NaOH) through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40-50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB) with 370-1110 GBq/μmol specific activity at EOB.

Full text of DOI:10.1016/j.bmcl.2015.07.061

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3831–3835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of [11C]CX-6258 as a new PET tracer for imaging of Pim
kinases in cancer
Min Wang a, Reynaldo Tzintzun a, Mingzhang Gao a, Zhidong Xu 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 Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding,
Hebei 071002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The reference standard CX-6258 {(E)-5-chloro-3-((5-(3-(4-methyl-1,4-diazepane-1-carbonyl)phenyl)
furan-2-yl)methylene)indolin-2-one, 4a} and its desmethylated precursor N-desmethyl-CX-6258
{(E)-3-((5-(3-(1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)-5-chloroindolin-2-one, 5} for
radiolabeling were synthesized from 5-bromo-2-furaldehyde and 3-carboxybenzeneboronic acid in 3
and 4 steps with 29–49% and 24–32% overall chemical yield, respectively. The target tracer [11C]CX-
Received 3 July 2015
Revised 16 July 2015
Accepted 21 July 2015
Available online 26 July 2015
6258
{(E)-5-chloro-3-((5-(3-(4-[11C]methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)
Keywords:
indolin-2-one, [11C]4a} was prepared from N-desmethyl-CX-6258 (5) with [11C]CH3OTf under basic
condition (2 N NaOH) through N-[11C]methylation and isolated by HPLC combined with solid-phase
extraction (SPE) in 40–50% radiochemical yield based on [11C]CO2 and decay corrected to end of
[
11C]CX-6258
Radiosynthesis
Positron emission tomography (PET)
Pim kinases
Cancer
bombardment (EOB) with 370–1110 GBq/
l
mol specific activity at EOB.
Ó 2015 Elsevier Ltd. All rights reserved.
Pim kinases (Provirus Integration site for Moloney murine leu-
kemia virus) are a family of serine/threonine kinases, including
three different members Pim1, Pim2 and Pim3.1–3 This family of
kinases regulates signaling pathways via Janus kinase (JAK)/signal
transducer and activator of transcription (STAT) pathway and plays
pivotal roles in cancer development and progression.4–6 Pim
kinases including Pim1, Pim2 and Pim3 are overexpressed not only
in hematologic malignancies but also in solid tumors, and have
become an attractive targets for therapeutics in cancer.7–10 Many
Pim kinases inhibitors have been discovered.11,12 Recently a potent,
selective, and orally efficacious pan–Pim kinases inhibitor called
CX-6258 has been developed by Cylene Pharmaceuticals, and the
IC50 of CX-6258 is 5, 25, and 16 nM for Pim1, Pim2, and Pim3,
respectively.13 Pim kinases have also become a promising target
for molecular imaging of Pim kinases-related cancers and image-
guided therapy using the biomedical imaging technique positron
emission tomography (PET). In our previous work, we have
developed a series of radiolabeled (carbon-11 and fluorine-18)
kinases. Here we report the design, synthesis and labeling of
[
11C]CX-6258 (Fig. 1).
The reference standard CX-6258 {(E)-5-chloro-3-((5-(3-(4-
methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)
indolin-2-one, 4a} and its [11C]-labeling precursor N-desmethyl-
CX-6258 {(E)-3-((5-(3-(1,4-diazepane-1-carbonyl)phenyl)furan-2-
yl)methylene)-5-chloroindolin-2-one, 5} were prepared based on
the reported method13 with modifications. As shown in
Scheme 1, Suzuki cross-coupling of 3-carboxybenzeneboronic acid
with 5-bromo-2-furaldehyde in the presence of aqueous 2 M
Na2CO3 using Pd(OAc)2 as catalyst in toluene/EtOH to give 3-(5-
formylfuran-2-yl)benzoic acid (1) in 89% yield.21 Compounds 2a
and 2b were achieved via amide bond coupling of acid 1 with
homopiperazines in the presence of N,N-diisopropylethylamine
(DIPEA) using O-(benzotriazo-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU) as coupling agent in N,N-dimethyl-
formamide (DMF) with 75% and 64% yield, respectively.22
Condensation of 5-chlorooxindole with aldehydes 2a and 2b using
piperidine as catalyst in EtOH afforded standard compound 4a and
N-Boc protected precursor 4b with 44% and 64% yield, respec-
tively.23 Compounds 4a and 4b could be obtained by reverse the
above two steps in much higher yields. Condensation of compound
1 with 5-chlorooxindole using piperidine as catalyst in EtOH to
provide acid 3 in 70% yield, which was coupled with homopiperazi-
nes using 1,10-carbonyldimidazole (CDI) as coupling agent in DMF
to provide 4a and 4b in 78% and 79% yield, respectively. The Boc
protein kinase (PK) inhibitors such as
31-7453),
11C]SB-216763, 11C]Enzastaurin ([11C]LY317615),
11C]GSK2126458, 2-[18F]GSK2126458 and 4-[18F]GSK2126458, as
[
11C]MKC-1 ([11C]Ro
[
[
[
shown in Figure 1, as new cancer imaging agents for PET to study
PK and PK inhibitors.14–20 In this ongoing study, we target Pim
⇑
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
http://dx.doi.org/10.1016/j.bmcl.2015.07.061
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

3832
M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3831–3835
H
N
H
N
O
O
O
O
O2N
N
N
O2N
N
N
11CH3
11CH3
CH3
CH3
[
11C]MKC-1 ([11C]Ro 31-7453)
H
N
O
O
H
N
N
N
O
O
11CH3
N
Cl
Cl
N
11CH3
N
11C]SB-216763
[
11C]Enzastaurin ([11C]LY317615)
[
N
N
N
N
N
N
N
H311CO
N
H3CO
O
N
H3CO
O
N
O
O
O
O
S
S
S
N
H
F
N
H
N
H
18F
F
F
18F
N
F
N
11C]GSK2126458
4-[18F]GSK2126458
2-[18F]GSK2126458
[
O
O
NH
N
O
Cl
N
H311C
[
11C]CX-6258
Figure 1. Radiolabeled PK inhibitors.
(HO)2B
CO2H
Pd(OAc)2, (C6H5)3P
CO2H
OHC
OHC
O
+
Br
O
Toluene/EtOH, 2 M Na2CO3
1
, 89%
CHO
O
O
R
HN
N
HN
O
Cl
O
HBTU, DIPEA, DMF
N
p
O
ip
e
r
i
d
i
n
e
,
E
t
O
H
NH
2a: R = CH3, 75%
N
N
O
2b
R
: R=Boc, 64%
Cl
N
R
N
Cl
R
HN
O
N
H
4a
(CX-6258): R = CH3, 44% (78%)
4b: R = Boc, 64% (79%)
O
O
F
M
D
.
I
D
C
piperidine, EtOH
NH
CO2H
Cl
3
, 70%
O
O
TFA, CH2Cl2
NH
N
O
Cl
N
H
5
(N-desmethyl-CX-6258), 66%
Scheme 1. Synthesis of the reference standard CX-6258 (4a) and its precursor N-desmethyl-CX-6258 (5).

M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3831–3835
3833
O
O
O
O
[
11C]CH3OTf, CH3CN
2 N NaOH
NH
NH
N
O
N
Cl
O
Cl
N
N
H
H311C
11C]4a ([11C]CX-6258), 40-50%
[
5
(N-desmethyl-CX-6258)
Scheme 2. Synthesis of the target tracer [11C]CX-6258.
group of 4b was removed with trifluoroacetic acid (TFA) in CH2Cl2
to afford the precursor 5 in 66% yield.
irradiation target system, and (2) carrier from the [11C]CH3OTf sys-
tem,
11C]-radiolabeled precursor or called 11C]-radiolabeled
[
[
Synthesis of the target tracer [11C]CX-6258 {(E)-5-chloro-3-
((5-(3-(4-[11C]methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-
yl)methylene)indolin-2-one, [11C]4a} is indicated in Scheme 2. The
desmethylated precursor 5 was labeled by [11C]methyl triflate
([11C]CH3OTf)24,25 through N-[11C]methylation26–28 at 80 °C under
basic condition (2 N NaOH) and isolated by a semi-preparative
reverse-phase (RP) high performance liquid chromatography
(HPLC) with a C-18 column and a solid-phase extraction (SPE) with
a disposable C-18 Plus Sep-Pak cartridge (a second purification or
isolation process)29–31 to produce the corresponding pure radiola-
beled compound [11C]4a in 40–50% radiochemical yield, decay
corrected to end of bombardment (EOB), based on [11C]CO2.
There are two nitrogen positions in the precursor 5 where the
N-[11C]methylation reaction could potentially happen. However,
the reactivity for N-[11C]methylation is different between these
two positions. The acidity of amine –NH is greater than
amide –NH, thus the deprotonization at –NH of amine is easier
than at –NH of amide under basic condition, the
N-[11C]methylation reaction would prefer to occur at amine
–NH, and the target tracer [11C]4a would be a major product.
methylating reagent. If we can eliminate 12C carrier-added as much
as possible, then we will be able to achieve higher SA. The [11C]-gas
target we used is the Siemens RDS-111 Eclipse cyclotron [11C]-gas
target. The technical trick to produce high SA [11C]CO2 is we will
usually do 2–3 times pre-burn with the same beam current and
short time like 10 min before production run. This pre-burn will
warm up the cyclotron and eliminate significant amount of 12C car-
rier-added in the cyclotron [11C]-gas target. The [11C]CH3OTf pro-
duction system we used is an Eckert & Ziegler Modular Lab C-11
Methyl Iodide/Triflate module, convenient gas phase bromination
of [11C]methane and production of [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.25
Chemical purity and radiochemical purity were determined by
analytical HPLC.38 The chemical purity of the precursor 5 and ref-
erence standard 4a was >93%. The radiochemical purity of the tar-
get tracer [11C]4a was >99% determined by radio-HPLC through
c-
ray (PIN diode) flow detector, and the chemical purity of [11C]4a
was >90% determined by RP HPLC through UV flow detector. A C-
18 Plus Sep-Pak cartridge instead of rotatory evaporation was used
for purification and isolation process to significantly improve the
chemical purity of the tracer solution.29–31,38 In this study, the
Sep-Pak purification further increased the chemical purity
>10%.29–31
[
11C]CH3OTf was used as the [11C]-radiolabeled precursor,
which is more reactive than another commonly used [11C]-methy-
lation reagent [11C]methyl iodide ([11C]CH3I)32, and thus, the radio-
chemical yield of [11C]4a was relatively higher. Basic condition
would help the N-[11C]methylation of precursor and significantly
increase the radiochemical yield of [11C]4a. Small amount of the
precursor (0.3–0.5 mg) was used for radiolabeling instead of com-
monly used large amount of the precursor (1.0–1.5 mg), which
improved the chemical purity of the final tracer solution.
However, the small amount of precursor could potentially decrease
the radiolabeling yield and increase the specific activity of the tar-
get tracer by decreasing the radiolabeling product as well as pro-
duct mass. In addition, in order to make more product
radioactivity, we also optimized the semi-preparative HPLC condi-
tions including column, mobile phase and flow rate to shorten the
retention time of [11C]4a to 8–9 min. Addition of NaHCO3 solution
to quench the radiolabeling reaction and to dilute the radiolabeling
mixture prior to the injection onto the semi-preparative HPLC
column for purification gave better separation of [11C]4a from its
desmethylated precursor 5.29–31,33
The experimental details and characterization data for com-
pounds 1–5 and for the tracer [11C]4a are given.39
In summary, a multi-step synthetic route with moderate to high
yields for the synthesis of the precursor N-desmethyl-CX-6258, ref-
erence standard CX-6258 and target tracer [11C]CX-6258 has been
developed.
[
An
automated
self-designed
multi-purpose
11C]-radiosynthesis module for the synthesis of [11C]CX-6258 has
been built, featuring the measurement of specific activity by
the on-the-fly technique. The radiosynthesis employed
N-[11C]methylation radiolabeling on amine nitrogen position of
the precursor. Radiolabeling procedures incorporated efficiently
with the most commonly used
[
[
11C]methylating agent,
11C]CH3OTf, produced by gas-phase production of [11C]methyl bro-
mide ([11C]CH3Br) from our laboratory. The target tracer was iso-
lated and purified by a semi-preparative RP HPLC combined with
SPE procedure in high radiochemical yield, radiochemical purity
and chemical purity, short overall synthesis time, and high specific
activity. These chemistry results warrant further biological evalua-
tion of [11C]CX-6258 as a new PET agent for imaging of Pim kinases
in cancer.
The radiosynthesis was performed in a home-built automated
multi-purpose [11C]-radiosynthesis module, allowing measure-
ment of specific radioactivity during synthesis.34–36 This [11C]-ra-
diosynthesis module includes the overall design of the reaction,
purification and reformulation capabilities of the prototype sys-
tem. In addition, [11C]-tracer specific activity (SA, GBq/
lmol at
EOB) can be automatically determined prior to product delivery
for compounds purified by the HPLC-portion of the system.36,37
Acknowledgments
The SA was in a range of 370–1110 GBq/
the
11C]-tracers synthesized by
11C]CH3OTf in our PET chemistry facility is depended on two
parts: (1) carrier from the cyclotron consisted of the [11C]-gas
l
[
mol at EOB. The SA for
[
11C]-methylation with
1H 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 Indianapolis
[

3834
M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3831–3835
was run using Analtech, silica gel UV 254 plates (20 ꢀ 20 cm2). 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
(IUPUI), which is supported by the United States National Science
Foundation (NSF) Major Research Instrumentation Program (MRI)
Grant CHE-0619254.
from
(Phenomenex) 5
70% 20 mM H3PO4; flow rate 2.0 mL/min; and UV (254 nm) and
diode) flow detectors. Semi-preparative HPLC was performed using a Prodigy
(Phenomenex) 5
m C-18 column, 12 nm, 10 ꢀ 250 mm; mobile phase 30%
CH3CN/70% 20 mM H3PO4; 6.0 mL/min flow rate; UV (254 nm) and -ray (PIN
diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from Waters
a
nitrogen source. Analytical HPLC was performed using
m C-18 column, 4.6 ꢀ 250 mm; mobile phase 30% CH3CN/
-ray (PIN
a Prodigy
References and notes
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l
m filter units were obtained
a
mixture of 3-carboxyben-
zeneboronic acid (2.0 g, 12.05 mmol), 5-bromo-2-furaldehyde (2.11 g,
12.05 mmol), Pd(OAc)2 (135.3 mg, 0.60 mmol) and triphenyl phosphine
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Na2CO3. After the reaction mixture was heated at reflux overnight, it was
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with EtOAc. The aqueous layer was acidified with aqueous 6 M HCl to pH 1–2.
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J = 7.5 Hz, 1H), 7.69–7.64 (m, 2H), 7.43 (d, J = 3.5 Hz, 1H). LC–MS (ESI, m/z):
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(c) 5-(3-(4-Methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-carbaldehyde (2a):
To a stirred solution of compound 1 (150 mg, 0.69 mmol) in anhydrous DMF
(4 mL) was added HBTU (394 mg, 1.04 mmol), DIPEA (0.36 mL, 2.08 mmol)
under N2 atmosphere. After the mixture was stirred at rt for 20 min, N-
methylhomopiperazine (0.13 mL, 1.04 mmol) was added. The reaction mixture
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J = 7.5 Hz, 1H), 7.30 (d, J = 6.5 Hz, 1H), 7.25 (d, J = 4.0 Hz, 1H), 6.81 (s, 1H), 3.72-
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7.39 (s, 1H), 7.29-7.25 (m, 2H), 6.88, 6.81 (s+s, 1H), 3.73-3.35 (m, 8H), 1.88 (br
m, 1H), 1.61 (br m, 1H), 1.39 (s, 9H). LC–MS (ESI, m/z): Calcd for C22H26N2O5Na
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27. Gao, M.; Shi, Z.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 23, 1953.
28. Wang, M.; Gao, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 23, 5259.
29. Gao, M.; Wang, M.; Mock, B. H.; Glick-Wilson, B. E.; Yoder, K. K.; Hutchins, G.
D.; Zheng, Q.-H. Appl. Radiat. Isot. 2010, 68, 1079.
30. Wang, M.; Gao, M.; Miller, K. D.; Zheng, Q.-H. Steroids 2011, 76, 1331.
31. Wang, M.; Gao, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2014, 24, 4455.
32. Allard, M.; Fouquet, E.; James, D.; Szlosek-Pinaudm, M. Curr. Med. Chem. 2008,
15, 235.
33. Gao, M.; Wang, M.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2008, 66,
1891.
(e) (E)-3-(5-((5-Chloro-2-oxoindolin-3-ylidene)methyl)furan-2-yl)benzoic acid
(3): To
a stirred suspension of compound 1 (1.5 g, 6.94 mmol) and 5-
chlorooxindole (1.4 g, 8.33 mmol) in anhydrous EtOH (20 mL) was added
piperidine (0.82 mL, 8.33 mmol) dropwise under N2 atmosphere. After the
reaction mixture was heated and stirred at 35 °C for 4 h, it was allowed to stir
at RT overnight. The solid was collected by filtration, washed with cold EtOH
and hexanes to afford compound 3 as an orange solid (1.78 g, 70%). 1H NMR
(DMSO-d6): d 13.19 (br s, 1H), 10.72 (s, 1H), 8.51 (s, 1H), 8.45 (s, 1H), 8.17 (d,
J = 6.5 Hz, 1H), 8.00 (d, J = 6.0 Hz, 1H), 7.68 (s, 1H), 7.49 (s, 2H), 7.41 (s, 1H),
7.32 (d, J = 7.0 Hz, 1H), 6.91 (d, J = 7.0 Hz, 1H). LC-MS (ESI, m/z): Calcd for
34. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd. Radiopharm. 2005, 48,
S225.
35. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd.
Radiopharm. 2005, 48, S224.
36. Wang, M.; Gao, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 965.
37. Gao, M.; Wang, M.; Green, M. A.; Hutchins, G. D.; Zheng, Q.-H. Bioorg. Med.
Chem. Lett. 1965, 2015, 25.
C
(f)
20H11ClNO4 ([MꢁH]ꢁ) 364.0; found 364.0.
(E)-5-Chloro-3-((5-(3-(4-methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-
yl)methylene)indolin-2-one (CX-6258, 4a): Method A. To a stirred suspension
of compound 2a (90 mg, 0.29 mmol) and 5-chlorooxindole (51 mg, 0.30 mmol)
in anhydrous EtOH (3 mL) was added piperidine (0.1 mL, 1.0 mmol) dropwise
under N2 atmosphere. After the reaction mixture was heated at reflux for 5 h,
the solvent was removed in vacuo. The residual was purified with preparative
TLC plate solid with CH2Cl2/MeOH (10:1) as eluent to afford compound 4a as
an orange solid (59 mg, 44%). 1H NMR (DMSO-d6): d 10.74 (s, 1H), 8.53 (s, 1H),
8.03 (d, J = 8.0 Hz, 1H), 7.94 (s, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.50 (s, 2H), 7.44–
7.42 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 3.64-3.63 (m, 2H),
3.41-3.38 (m, 2H), 2.65 (br m, 1H), 2.55 (br m, 1H), 2.51–2.47 (m, 2H), 2.29,
2.18 (s+s, 3H), 1.88 (br m, 1H), 1.73 (br m, 1H). LC–MS (ESI, m/z): Calcd for
38. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671.
39. (a) General: All commercial reagents and solvents were purchased from Sigma-
Aldrich and Fisher Scientific, and used without further purification.
[
11C]CH3OTf was prepared according to literature procedure.25 Melting
a
points were determined on a MEL-TEMP II capillary tube apparatus and were
uncorrected. 1H NMR spectra were recorded at 500 MHz 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). Liquid chromatography-
mass spectra (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.
Chromatographic solvent proportions are indicated as volume:volume ratio.
Thin-layer chromatography (TLC) was run using Analtech silica gel GF254
uniplates (5 ꢀ 10 cm2). Plates were visualized under UV light. Preparative TLC
C
26H25ClN3O3 ([M+H]+) 462.2, found 462.2. Method B. To a stirred solution of
compound 3 (150 mg, 0.41 mmol) in anhydrous DMF (4 mL) was added CDI
(133 mg, 0.82 mmol) under N2 atmosphere. After the mixture was stirred at rt
overnight, N-methylhomopiperazine (0.13 mL, 1.04 mmol) was added. The
reaction mixture was stirred for 24 h. Water (12 mL) was added to quench the
reaction, and the mixture was stirred for 1 h. The precipitate was collected by
filtration, washed with water, dried in vacuo. The crude product was purified
with preparative TLC plates with CH2Cl2/MeOH (25:3) as eluent to afford

M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3831–3835
3835
compound 4a as an orange solid (148.4 mg, 78%). Analytical data was identical
with Method A.
base was prepared by addition of compound 5 TFA salt in water and basified
with NH4OH to pH 10. The mixture was extracted with CH2Cl2. The combined
organic layer was washed with water, brine and dried over anhydrous Na2SO4,
filtered and concentrated in vacuo to afford an orange solid for labelling.
(i) (E)-5-Chloro-3-((5-(3-(4-[11C]methyl-1,4-diazepane-1-carbonyl)phenyl)furan-
2-yl)methylene)indolin-2-one ([11C]CX-6258, [11C]4a): [11C]CO2 was produced
(g) tert-Butyl (E)-4-(3-(5-((5-chloro-2-oxoindolin-3-ylidene)methyl)furan-2-
yl)benzoyl)-1,4-diazepane-1-carboxylate (4b): Method A. To a stirred suspension
of compound 2b (100 mg, 0.25 mmol) and 5-chlorooxindole (44 mg,
0.26 mmol) in anhydrous EtOH (2.5 mL) was added piperidine (0.1 mL,
1.0 mmol) dropwise under N2 atmosphere. After the reaction mixture was
heated at reflux for 5 h, the solvent was removed in vacuo. The residual was
purified with preparative TLC plate solid with CH2Cl2/MeOH (50:3) as eluent to
afford compound 4b as an orange solid (137 mg, 64%). 1H NMR (CDCl3): d 8.77
(s, 1H), 8.58, 8.55 (s+s, 1H), 7.82 (d, J = 7.0 Hz, 1H), 7.51 (quin, J = 7.5 Hz, 1H),
7.38–7.34 (m, 2H), 7.17 (dd, J = 8.0, 17.0 Hz, 1H), 6.82, 6.77 (d+d, J = 8.0, 8.0 Hz,
1H), 3.83 (br m, 1H), 3.74 (t, J = 5.5 Hz, 1H), 3.66 (br m, 1H), 3.57-3.39 (m, 5H),
1.99 (br m, 1H), 1.73 (m, 1H), 1.50 (s, 9H). LC–MS (ESI, m/z): Calcd for
by the 14N(p, 11C nuclear reaction in the small volume (9.5 cm3) aluminum
a)
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
beam current and 30 min on target. The production run produced approxi-
mately 45.5 GBq of small reaction vial (5 mL), the
11C]CO2 at EOB. In
lA
[
a
precursor 5 (0.3–0.5 mg) was dissolved in DMSO (400 lL). To this solution was
added NaH (1 mg). No carrier-added (high specific activity) [11C]CH3OTf that
was produced by the gas-phase production method25 from [11C]CO2 through
C
30H29ClN3O5 ([MꢁH]ꢁ) 546.2, found 546.2. Method B. To a stirred solution of
compound 3 (200 mg, 0.55 mmol) in anhydrous DMF (5 mL) was added CDI
(178 mg, 1.1 mmol) under N2 atmosphere. After the mixture was stirred at rt
overnight, 1-Boc-hexahydro-1,4-diazapine (0.27 mL, 1.38 mmol) was added.
The reaction mixture was stirred for 24 h. Water (15 mL) was added to quench
the reaction, and the mixture was stirred for 1 h. The precipitate was collected
by filtration, washed with water, dried in vacuo. The crude product was
purified with preparative TLC plates with CH2Cl2/MeOH (50:3) as eluent to
afford compound 4b as an orange solid (300 mg, 79%). Analytical data was
identical with Method A.
[
11C]CH4 and [11C]CH3Br with silver triflate (AgOTf) column was passed into
the reaction vial at rt, 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 solution (0.1 M, 1 mL), and
injected onto the semi-preparative RP 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 ꢀ 4). The cartridge
was eluted with EtOH (1 mL ꢀ 2), followed by 10 mL saline, to release [11C]4a.
The eluted product was then sterile-filtered through a sterile vented Millex-FG
(h)
(E)-3-((5-(3-(1,4-Diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)-5-
chloroindolin-2-one (N-desmethyl-CX-6258, 5): To a stirred solution of com-
pound 4b (80 mg, 0.15 mmol) in CH2Cl2 (1 mL) was added TFA (0.2 mL)
dropwise at 0 °C. After the reaction mixture was stirred for 4 h, the solvent was
removed in vacuo. The crude product was washed with Et2O to afford
compound 5 TFA salt as an orange solid (54 mg, 66%). 1H NMR (DMSO-d6): d
10.76 (s, 1H), 9.00 (s, 2H), 8.54 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.97 (s, 1H), 7.66
(t, J = 7.0 Hz, 1H), 7.55–7.42 (m, 2H), 7.34 (d, J = 7.5 Hz, 1H), 6.93 (d, J = 8.0 Hz,
1H), 3.86-3.63 (m, 3H), 3.45-3.44 (m, 2H), 3.25 (m, 3H), 2.07–1.95 (m, 2H). LC–
MS (ESI, m/z): Calcd for C25H23ClN3O3 ([M+H]+) 448.1, found 448.1. The free
0.2 lm filter, and collected into a sterile vial. Total radioactivity (4.6-8.2 GBq)
was assayed and total volume (10–11 mL) was noted for tracer dose dispens-
ing. The overall synthesis, purification and formulation time was 30–40 min
from EOB. Retention times in the analytical HPLC system were: tR 5 = 3.38 min,
t
R 4a = 5.51 min, tR
HPLC system were: tR 5 = 5.83 min, tR 4a = 8.67 min, tR
radiochemical yield of [11C]4a was 40–50% decay corrected to EOB, based on
11C]CO2.
[
11C]4a = 5.64 min. Retention times in the semi-preparative
[
11C]4a = 8.88 min. The
[