Synthesis of carbon-11-labeled CK1 inhibitors as new potential PET radiotracers for imaging of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2018.05.053

The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-methoxybenzamide (5c), and their corresponding desmethylated precursors 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b), were synthesized from 5-amino-2,2-difluoro-1,3-benzodioxole and 3-substituted benzoic acids in 5 and 6 steps with 33% and 11%, 30% and 7% overall chemical yield, respectively. Carbon-11-labeled casein kinase 1 (CK1) inhibitors, [¹¹C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([¹¹C]5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-[¹¹C]methoxybenzamide ([¹¹C]5c), were prepared from their O-desmethylated precursor 6a or 6b with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–45% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370–740 GBq/μmol with a total synthesis time of ～40-min from EOB.

Full text of DOI:10.1016/j.bmcl.2018.05.053

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of carbon-11-labeled CK1 inhibitors as new potential PET
radiotracers for imaging of Alzheimer’s disease
Mingzhang Gao, Min Wang, Qi-Huang Zheng⁎
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carba-
moyl)benzoate (5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-methox-
ybenzamide (5c), and their corresponding desmethylated precursors 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]
benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo
[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b), were synthesized from 5-amino-2,2-difluoro-1,3-benzodioxole
and 3-substituted benzoic acids in 5 and 6 steps with 33% and 11%, 30% and 7% overall chemical yield,
respectively. Carbon-11-labeled casein kinase 1 (CK1) inhibitors, [11C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo
[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([11C]5a) and N-(2,2-difluoro-5H-[1,3]dioxolo
[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-[11C]methoxybenzamide ([11C]5c), were prepared from their O-des-
methylated precursor 6a or 6b with [11C]CH3OTf through O-[11C]methylation and isolated by HPLC combined
with SPE in 40–45% radiochemical yield, based on [11C]CO2 and decay corrected to end of bombardment (EOB).
The radiochemical purity was > 99%, and the molar activity (MA) at EOB was 370–740 GBq/μmol with a total
synthesis time of ∼40-min from EOB.
Casein kinase 1 (CK1)
Carbon-11-labeled CK1 inhibitors
Radiosynthesis
Positron emission tomography (PET)
Alzheimer’s disease (AD)
Casein kinases (CK) are serine/threonine-specific enzymes and can
be divided two subtypes: casein kinase 1 (CK1) and casein kinase 2
(CK2).1 CK1 contains at least seven isoforms (α, β, γ1, γ2, γ3, δ and ε)
expressed in eukaryotic organisms, CK1 is involved in various cellular
processes including membrane trafficking, circadian rhythm, cell cycle
progression, chromosome segregation, apoptosis and cellular differ-
entiation, and deregulation of CK1 activity is linked to several patho-
logical disorders and diseases like cancer, neurodegenerative diseases
such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyo-
trophic lateral sclerosis (ALS), and inflammatory disorders.2,3 The
overexpression of CK1 has been described in the human AD brain, since
CK1 leads to an increase in amyloid-β (Aβ) peptide production, and also
participates in the tau fibrillization reaction pathway through phos-
phorylation of tau.4,5 CK1 has become an interesting therapeutic target
for AD where an urgent need for effective treatment exists, because it
opens the door for the use of CK1 inhibitors as novel therapeutic ap-
proaches for AD.6 CK1 inhibitors can prevent Aβ formation and reverse
tau hyperphosphorylation in AD, and have been used to treat neuro-
degenerative disorders including AD.4,5,7 Recently a new series of di-
fluoro-dioxolo-benzoimidazol-benzamides have been developed as po-
tent CK1 inhibitors with nanomolar inhibitory activity, these
compounds exhibited significant inhibitory effects on several tumor cell
lines, and their in vitro biological data suggested they can be as ther-
apeutics for AD as well.8
AD is a complicated neurodegenerative disease in the central ner-
vous system (CNS), the cause of AD remains unclear, and so far no any
effective treatment strategy is approved for preventing, curing and
slowing the progress of AD.9 To discover more effective treatments,
more accurate diagnostic tools are crucial to reveal new therapeutic
targets.10 New noninvasive diagnostic imaging modalities for AD are
really needed, both to detect and monitor the evolution of this disease,
and to evaluate the efficacy of treatments.11 Advanced biomedical
imaging technique positron emission tomography (PET) is one of the
most widespread imaging techniques for AD, and significant progress
has been made to develop PET agents for two key neuropathological
substrates of AD: Aβ plaques and tau neurofibrillary tangles (NFTs).12,13
The representative PET Aβ and tau tracers [11C]PIB and [18F]Amyvid
([18F]AV-45);14,15 11C]PBB3 and [18F]T807 ([18F]AV-1451)16,17 are
[
listed in Fig. 1. The development of PET imaging probes for in vivo
detection of Alzheimer’s brains is critical for early and accurate diag-
nosis and for the successful discovery of AD therapies.18 The success
and limitations of Aβ imaging and tau imaging have spurred efforts
worldwide to develop new selective PET tracers for different imaging
targets. CK1 has emerged as a new molecular imaging target of AD,19
⁎
Corresponding author.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
https://doi.org/10.1016/j.bmcl.2018.05.053
Received 5 April 2018; Received in revised form 27 May 2018; Accepted 28 May 2018
0960-894X/©2018PublishedbyElsevierLtd.
Pleasecitethisarticleas:Gao,M.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.05.053

M. Gao et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of reference standards 5a; 5c and O-desmethylated pre-
cursors 6a; 6b. Reaction reagents, conditions and yields: (i) acetic anhydride,
toluene, 100 °C, 92%. (ii) NO2BF4, CH2Cl2, 69%. (iii) hydrogen, Pd/C, me-
thanol; (iv) cyanogen bromide, methanol, room temperature (RT), 40 h, 82%.
(v) 3-substitutent-benzoic acid, HBTU, DIPEA, 18–63%. (vi) for 6a, KOH, me-
thanol, 93%; for 6b, Me2S, BF3.Et2O, CH2Cl2, 65%.
to provide the key intermediate amino 4 in 82% yield. The catalyst
change in hydrogenation also improved the yield. Then the amino 4
was reacted with several 3-substituted benzoic acids under the catalysis
of
N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexa-
Fig. 1. PET radiotracers for imaging of AD.
fluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) to
afford the standard compounds 5a and 5c in 63% and 21% yield, re-
spectively. A protected benzamide 5b was also synthesized in 18%
yield. Compound 5a was hydrolyzed in methanol solution of KOH to
yield its acid precursor 6a in 93% yield. Compound 5b was converted to
O-desmethylated precursor 6b for compound 5c through the depro-
tecting reaction of benzyl group employing boron trifluoride diethyl
etherate (BF3·Et2O) and dimethyl sulfide (Me2S) in 65% yield. This
deprotective reagent system was found to be better than other depro-
tective reagent system like H2 and Pd/C, which is easy to result in
byproduct formation and lower yield.
but so far no any carbon-11 or fluorine-18 labeled CK1 inhibitors as PET
radiotracers for imaging of CK1 were reported. We are interested in the
development of new PET AD imaging agents, and a series of enzyme- or
receptor-based PET agents have been developed in this laboratory. In
our previous work, we have targeted the enzyme glycogen synthase
kinase-3 (GSK-3) and developed carbon-11-labeled GSK-3 in-
hibitors;20,21 and we have also targeted serotonin (5-hydro-
xytryptamine) 6 receptor (5-HT6R) and developed carbon-11-labeled 5-
HT6R antagonists,22 as PET radiotracers for AD imaging (Fig. 1). In this
ongoing study, we first target CK1, which is a novel and attractive
molecular target for treatment and PET imaging of AD.19 Here, we re-
port the design, synthesis and labeling of carbon-11-labeled CK1 in-
hibitors, [11C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo
[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([11C]5a) and N-(2,2-di-
fluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-[11C]
methoxybenzamide ([11C]5c), as new potential PET radiotracers for
imaging of AD, for the first time. The basic evaluations of the radio-
tracers including lipophilicity and stability are presented as well.
The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo
[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (5a) and N-
(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-
methoxybenzamide (5c), and their corresponding desmethylated pre-
Synthesis of the target tracers [11C]5a and [11C]5c is indicated in
Scheme 2. O-Desmethylated precursor 6a or 6b underwent O-[11C]
methylation23,24 using the reactive [11C]methylating agent [11C]methyl
triflate ([11C]CH3OTf)25,26 in acetonitrile at 80 °C under basic condi-
tions (2N NaOH). The product was isolated by semi-preparative re-
verse-phase (RP) high performance liquid chromatography (HPLC) with
a C-18 column, and then concentrated by solid-phase extraction
(SPE)27,28 with a disposable C-18 Light Sep-Pak cartridge to produce
the corresponding pure radiolabeled compound [11C]5a or [11C]5c in
40–45% radiochemical yield, decay corrected to end of bombardment
(EOB), based on [11C]CO2.
The radiosynthesis was performed in a home-built automated multi-
purpose [11C]-radiosynthesis module.29–31 Our radiosynthesis module
facilitated the overall design of the reaction, purification and re-
cursors
3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imi-
dazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]
dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide
(6b), were synthesized as shown in Scheme 1, according to the litera-
ture method8 with modifications. The commercially available starting
material 5-amino-2,2-difluoro-1,3-benzodioxole was treated with acetic
anhydride in toluene to obtain acetamide 1 in 92% yield. Compound 1
was then converted to the intermediate 2 through a concurrent nitra-
tion and deprotection with nitronium tetrafluoroborate (NO2BF4) in
69% yield. In comparison with the reported method,8 the use of the
nitration reagent NO2BF4 simplified the reaction steps, combining ni-
tration reaction and deprotecting reaction into one step, and improved
the reaction yield. The nitro compound 2 was reduced through hy-
drogenation using H2 and Pd/C as catalyst instead of Raney Nickel
reported in the literature8 to give the intermediate 3 containing two
amino groups, which was subsequently reacted with cyanogen bromide
formulation capabilities in
a fashion suitable for adaptation to
Scheme 2. Synthesis of target tracers [11C]5a and [11C]5c. Reaction reagents,
conditions and yields: (i) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 °C, 3 min; HPLC-
SPE, 40–45%.
2

M. Gao et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
preparation of human doses. The radiosynthesis includes three stages:
Table 1
Log P and CLog P values of carbon-11-labeled CK1 inhibitors [11C]5a and [11C]
5c in comparison with [11C]PIB, [18F]Amyvid, [11C]PBB3 and [18F]T807.
1) labeling reaction; 2) purification; and 3) formulation. More reactive
[
11C]CH3OTf, instead of commonly used
[
11C]methyl iodide
([11C]CH3I),32 was used in O-[11C]methylation to improve radio-
chemical yield of [11C]5a and [11C]5c. The Eckert & Ziegler Modular
Lab C-11 Methyl Iodide/Triflate module in our facility can produce
Compound
Log P
CLog P
[
[
[
[
[
[
11C]5a
3.60
3.66
3.41
3.16
4.09
2.25
5.09
5.12
3.99
3.91
4.05
3.18
11C]5c
[
11C]methylating agent either [11C]CH3OTf or [11C]CH3I ([11C]CH3Br
11C]PIB
18F]Amyvid
11C]PBB3
18F]T807
passed through
a NaI column). The direct comparison between
[
11C]CH3OTf and [11C]CH3I confirmed the result that the labeling yield
was improved from 30 to 35% to 40–45%. The labeling reaction was
conducted using a V-vial method. Addition of aqueous NaHCO3 to
quench the radiolabeling reaction and to dilute the radiolabeling mix-
ture prior to the injection onto the semi-preparative HPLC column for
purification gave better separation of [11C]5a or [11C]5c from its O-
desmethylated precursor 6a or 6b. Both Sep-Pak trap/release and ro-
tatory evaporation are available for formulation in our multi-purpose
biological activities of a wide variety of organic compounds.36,38 Log P
provides an assessment of lipophilicity that often correlates with a
compound’s ability to penetrate the blood brain barrier (BBB). Log P
can be determined experimentally by liquid-liquid extraction and by
HPLC38 and can also be theoretically calculated from parameters re-
lated to the structure of molecules. We obtained Log P and calculated
Log P (CLog P) values of carbon-11-labeled CK1 inhibitors [11C]5a and
[
11C]-radiosynthesis module, and we used Sep-Pak method instead of
rotatory evaporation for formulation to improve the chemical purity of
radiolabeled product [11C]5a or [11C]5c. The direct comparison be-
tween Sep-Pak method and rotatory evaporation confirmed the result
that the chemical purity of radiolabeled product was improved
from < 90% to > 90%. In addition, a C18 Light Sep-Pak to replace a
C18 Plus Sep-Pak allowed final product formulation with ≤5%
ethanol.33 Overall, it took ∼40 min for synthesis, purification and dose
formulation.
[
[
11C]5c in comparison with [11C]PIB, [18F]Amyvid, [11C]PBB3 and
18F]T807 (Fig. 1) from ChemDraw Professional 15.1 (ChemOffice),
and the data are listed in Table 1. Log P data of [11C]5a and [11C]5c
(3.60–3.66) are in the range of Log P data of [11C]PIB, [18F]Amyvid,
[
11C]PBB3 and [18F]T807 (2.25–4.09), which are PET AD imaging
agents in clinical evaluation. These data suggest [11C]5a and [11C]5c
have appropriate lipophilicity to pass the BBB for brain uptake.
The stability of the labeled tracers [11C]5a and [11C]5c was eval-
uated by analytical HPLC from EOS up to 3 h, one injection of the tracer
solution in EtOH/saline onto HPLC column per hour. The HPLC chro-
matograms showed [11C]5a and [11C]5c were stable without decom-
position.
Our module is designed to allow in-process measurement of [11C]-
tracer molar activity (MA, GBq/μmol at EOB) using a radiation detector
with a UV detector at the outlet of the HPLC-portion of the system.31 In
the HPLC chromatogram, peak analysis of the chromatographic data
utilized PeakSimple software (SRI Instruments, Las Vegas, NV). Im-
mediately 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 – mCi at EOB by comparison to a
reference calibration curve previously constructed using the same de-
tector, loop column, mobile phase 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 –
mCi) by the total mass peak (in nmoles) gives MA at EOB in GBq – Ci/
μmol. For the reported syntheses, product MA was in a range of
370–740 GBq/μmol at EOB. The factors that affect the EOB MA sig-
nificantly to lead to such a wide range have been discussed in our
previous works.34–36 The general methods to increase MA have been
described as well, and the MA of our [11C]-tracers is significantly im-
proved.34–36 The ‘wide range’ of MA we reported is for the same [11C]-
tracer produced in different days, because very different [11C]-target
and [11C]-radiosynthesis unit situations would make MA in a wide
range. For a [11C]-tracer produced in the same day, the MA of the same
tracer in different production runs will be in a small range, because
The experimental details and characterization data for compounds
1–6 and for the tracers [11C]5a and [11C]5c are given.39
In summary, synthetic routes with moderate to high yields have
been developed to produce difluoro-dioxolo-benzoimidazol-benzamides
including reference standards 5a and 5c, and O-desmethylated pre-
cursors 6a and 6b, and carbon-11-labeled difluoro-dioxolo-benzoimi-
dazol-benzamides target tracers
[ [
11C]5a and 11C]5c. The radio-
synthesis employed
[
11C]CH3OTf for O-[11C]methylation at O-
desmethylated precursor, followed by product purification and isola-
tion using a semi-preparative RP HPLC combined with SPE. [11C]5a and
[
11C]5c were obtained in high radiochemical yield, radiochemical
purity and chemical purity, with a reasonable short overall synthesis
time, and high molar activity. This will facilitate studies to evaluate
carbon-11-labeled CK1 inhibitors [11C]5a and [11C]5c as new candi-
date PET radiotracers for imaging of AD.
Acknowledgments
This work was partially supported by Indiana University
[
11C]-target and [11C]-radiosynthesis unit would not be much different
Department of Radiology and Imaging Sciences in the United States. 1
H
in the same day. Likewise, the methods to minimize such wide range of
MA from practice perspective have been provided in our previous
works.34–36 At the end of synthesis (EOS), the MA of [11C]-tracer was
determined again by analytical RP HPLC, calculated, decay corrected to
EOB, and based on [11C]CO2, which was in agreement with the ‘on line’
determined value. In this work, semi-preparative HPLC was used for
purification, thus the MA of [11C]-tracer was assessed by both semi-
preparative HPLC (during synthesis) and analytical HPLC (EOS).
Chemical purity and radiochemical purity were determined by
analytical HPLC.37 The chemical purity of the precursor and reference
standard was > 93%. The radiochemical purity of the target tracer
was > 99% determined by radio-HPLC through γ-ray (PIN diode) flow
detector, and the chemical purity of the target tracer was > 90% de-
termined by reversed-phase HPLC through UV flow detector.
NMR and 13C NMR spectra were recorded at 500 and 125 MHz, re-
spectively, on a Bruker Avance II 500 MHz NMR spectrometer in the
Department of Chemistry and Chemical Biology at Indiana University
Purdue University Indianapolis (IUPUI), which is supported by the
United States National Science Foundation (NSF) Major Research
Instrumentation Program (MRI) grant CHE-0619254.
References
1. Schittek B, Sinnberg T. Biological functions of casein kinase 1 isoforms and putative
roles in tumorigenesis. Mol Cancer. 2014;13:231.
2. Vielhaber E, Virshup DM. Casein kinase I: from obscurity to center stage. IUBMB Life.
2001;51:73–78.
3. Rudrabhatla P. Regulation of neuronal cytoskeletal protein phosphorylation in neu-
rodegenerative diseases. J Alzheimer’s Dis. 2014;41:671–684.
4. Flajolet M, He G, Heiman M, Lin A, Nairn AC, Greengard P. Regulation of Alzheimer’s
The octanol-water partition coefficient (commonly expressed as Log
P) is an important physical parameter directly correlated with the
disease amyloid-beta formation by casein kinase I. Proc Natl Acad Sci USA.
3

M. Gao et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
2007;104:4159–4164.
5. Li G, Yin H, Kuret J. Casein kinase 1 delta phosphorylates tau and disrupts its binding
38. Liu X, Wang JQ, Zheng Q-H. Lipophilicity coefficients of potential tumor imaging
agents, positron-labeled O6-benzylguanine derivatives. Biomed Chromatogr.
to microtubules. J Biol Chem. 2004;279:15938–15945.
2005;19:379–384.
6. Perez DI, Gil C, Martinez A. Protein kinases CK1 and CK2 as new targets for neuro-
degenerative diseases. Med Res Rev. 2011;31:924–954.
7. Benek O, Hroch L, Aitken L, et al. 1-(Benzo[d]thiazol-2-yl)-3-phenylureas as dual
inhibitors of casein kinase 1 and ABAD enzymes for treatment of neurodegenerative
disorders. J Enzyme Inhib Med Chem. 2018;33:665–670.
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 a literature procedure.26 Melting points were determined on WRR
apparatus and were uncorrected. 1H and 13C NMR spectra were recorded on a Bruker
Avance II 500 MHz NMR Fourier transform spectrometer at 500 and 125 MHz, respec-
tively. Chemical shifts (δ) are reported in parts per million (ppm) relative to an internal
standard tetramethylsilane (TMS, 0.0) (1H NMR) and to the solvent signal (13C NMR), and
coupling constants (J) are 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 po-
sitive-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 RP HPLC was performed using a Prodigy (Phenomenex) 5 μm C-18 column,
4.6 × 250 mm; mobile phase 60%CH3CN/40% H2O; flow rate 1.3 mL/min; UV (254 nm)
and γ-ray (PIN diode) flow detectors. Semi-preparative RP HPLC was performed using a
Prodigy (Phenomenex) 5 μm C-18 column, 10 × 250 mm; mobile phase 60%CH3CN/40%
H2O; flow rate 5 mL/min; UV (254 nm) and γ-ray (PIN diode) flow detectors. C18 Light
Sep-Pak cartridges were obtained from Waters Corporation (Milford, MA). Sterile Millex-
FG 0.2 μm filter units were obtained from Millipore Corporation (Bedford, MA). (b). N-
(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)acetamide (1): A solution of 5-amino-2,2-difluoro-
1,3-benzodioxole (14.4 g, 83.2 mmol) in dry toluene (230 mL) and acetic anhydride
(9.76 g, 95.7 mmol, 1.15 equiv) was stirred at 100 °C for 3 h. The solvent was removed
under reduced pressure, then the crude product was dissolved in 80 mL of methanol to
remove traces of acetic anhydride. The solvent was subsequently evaporated. The crude
product was recrystallized from toluene, and the resulting product was filtered off and
dried to give a beige crystal 1 (16.5 g, 92%), Rf = 0.25 (1:2 EtOAc/Hexanes), mp
140–142 °C. 1H NMR (DMSO-d6): 2.05 (s, 3H, CH3), 7.21 (dd, J = 2.0, 8.5 Hz, 1H,
Ph−H), 7.31 (d, J = 8.5 Hz, 1H, Ph−H), 7.75 (d, J = 2.0 Hz, 1H, Ph−H), 10.14 (s, 1H,
NH). MS (ESI): 216 ([M+H]+, 100%); MS (ESI): 214 ([M−H]−, 100%). (c). 2,2-Difluoro-
6-nitrobenzo[d][1,3]dioxol-5-amine (2): Compound 1 (15.6 g, 72.5 mmol) was dissolved in
dichloromethane (250 mL). To the resulting mixture, nitronium tetrafluoroborate solu-
tion (174 mL, 0.5 M in sulfolane) was added dropwise at 0 °C. After addition, the reaction
mixture was allowed to RT and stirred for 24 h. Then the reaction mixture was evaporated
to remove dichloromethane, and the resulting mixture was added with water, extracted
with EtOAc, washed with water two times, and dried over Na2SO4. The mixture was
filtered, and filtrate was evaporated in vacuo. The resultant residue was purified by
column chromatography on silica gel with eluent (2:98 to 20:80 EtOAc/hexanes) to give a
light beige solid 2 (10.9 g, 69%), Rf = 0.73 (CH2Cl2), mp 138–140 °C. 1H NMR (DMSO-
8. Richter J, Bischof J, Zaja M, et al. Difluoro-dioxolo-benzoimidazol-benzamides as
potent inhibitors of CK1δ and ε with nanomolar inhibitory activity on cancer cell
proliferation. J Med Chem. 2014;57:7933–7946.
9. Wang M, Gao M, Zheng Q-H. The first synthesis of [11C]J147, a new potential PET
agent for imaging of Alzheimer’s disease. Bioorg Med Chem Lett. 2013;23:524–527.
10. Pietrzak K, Czarnecka K, Mikiciuk-Olasik E, Szymanski P. New perspectives of
Alzheimer disease diagnosis – the most popular and future methods. Med Chem.
2018;14:34–43.
11. Mach RH. New targets for the development of PET tracers for imaging neurode-
generation in Alzheimer disease. J Nucl Med. 2014;55:1221–1224.
12. Hall B, Mak E, Cervenka S, Aigbirhio FI, Rowe JB, O’Brien JT. In vivo tau PET
imaging in dementia: pathophysiology, radiotracer quantification, and a systematic
review of clinical findings. Ageing Res Rev. 2017;36:50–63.
13. Oukoloff K, Cieslikiewicz-Bouet M, Chao S, et al. PET and SPECT radiotracers for
Alzheimer’s disease. Curr Med Chem. 2015;22:3278–3304.
14. Klunk WE, Engler H, Nordberg A, et al. Imaging the pathology of Alzheimer’s disease:
amyloid-imaging with positron emission tomography. Neuroimaging Clin N Am.
2003;13:781–789.
15. Carpenter Jr AP, Pontecorvo MJ, Hefti FF, Skovronsky DM. The use of the ex-
ploratory IND in the evaluation and development of 18F-PET radiopharmaceuticals
for amyloid imaging in the brain: a review of one company’s experience. Q J Nucl
Med Mol Imaging. 2009;53:387–393.
16. Hashimoto H, Kawamura K, Igarashi N, et al. Radiosynthesis, photoisomerization,
biodistribution, and metabolite analysis of 11C-PBB3 as a clinically useful PET probe
for imaging of tau pathology. J Nucl Med. 2014;55:1532–1538.
17. Chien DT, Bahri S, Szardenings AK, et al. Early clinical PET imaging results with the
novel PHF-tau radioligand [F-18]-T807. J Alzheimers Dis. 2013;34:457–468.
18. Okamura N, Harada R, Furukawa K, et al. Advances in the development of tau PET
radiotracers and their clinical applications. Ageing Res Rev. 2016;30:107–113.
19. Wager TT, Galatsis P, Chandrasekaran RY, et al. Identification and profiling of a
selective and brain penetrant radioligand for in vivo target occupancy measurement
of casein kinase 1 (CK1) inhibitors. ACS Chem Neurosci. 2017;8:1995–2004.
20. Wang M, Gao M, Miller KD, Sledge GW, Hutchins GD, Zheng Q-H. The first synthesis
of [11C]SB-216763, a new potential PET agent for imaging of glycogen synthase
kinase-3 (GSK-3). Bioorg Med Chem Lett. 2011;21:245–249.
21. Gao M, Wang M, Zheng Q-H. Synthesis of carbon-11-labeled isonicotinamides as new
potential PET agents for imaging of GSK-3 enzyme in Alzheimer’s disease. Bioorg Med
Chem Lett. 2017;27:740–743.
22. Wang X, Dong F, Miao C, et al. Synthesis of carbon-11-labeled 5-HT6R antagonists as
new potential PET radioligands for imaging of Alzheimer’s disease. Bioorg Med Chem
Lett. 2018;28:1836–1841.
d6): 6.94 (s, 1H, Ph−H), 7.79 (s, 2H, NH2), 7.94 (s, 1H, Ph−H). MS (ESI): 219 ([M+H]+
,
3%); MS (ESI): 217 ([M−H]−, 100%). (d). 2,2-Difluorobenzo[d][1,3]dioxole-5,6-diamine
(3): Compound 2 (5.0 g, 22.9 mmol) was dissolved in methanol (120 mL) and hydro-
genated under hydrogen atmosphere (55 psi) with Pd/C (300–500 mg) as a catalyst for
4 h. The reaction mixture was filtered over Celite 545 and washed with methanol. The
solvent was evaporated in vacuo to obtain a purple solid 3, which was directly used for
next step without further purification because this compound was unstable on silica gel
23. Wang M, Gao M, Xu Z, Zheng Q-H. Synthesis of [11C]HG-10-102-01 as a new po-
tential PET agent for imaging of LRRK2 enzyme in Parkinson’s disease. Bioorg Med
Chem Lett. 2017;27:1351–1355.
24. Gao M, Wang M, Meyer JA, et al. Synthesis and preliminary biological evaluation of
[
11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for
the fractalkine receptor (CX3CR1). Bioorg Med Chem Lett. 2017;27:2727–2730.
25. Jewett DM. A simple synthesis of [11C]methyl triflate. Int J Rad Appl Instrum A.
1992;43:1383–1385.
column. Rf = 0.34 (CH2Cl2). MS (ESI): 189 ([M+H]+, 100%); MS (ESI): 187 ([M−H]−
45%). (e). 2,2-Difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-amine (4): The
crude product 3 (0.94 g, 5.0 mmol) was dissolved in dry methanol, cyanogen bromide
(795 mg, 7.5 mmol) was added. The reaction mixture was stirred at RT for 40 h. The
reaction mixture was concentrated, and the residue was purified by column chromato-
graphy on silica gel with eluent (2:98 to 10:90 MeOH/CH2Cl2) to give a brown solid 4
(875 mg, 82%), Rf = 0.27 (1:19 MeOH/CH2Cl2), mp 135–137 °C. 1H NMR (DMSO-d6):
,
26. Mock BH, Mulholland GK, Vavrek MT. Convenient gas phase bromination of [11C]
methane and production of [11C]methyl triflate. Nucl Med Biol. 1999;26:467–471.
27. Wang M, Gao M, Miller KD, Zheng Q-H. Synthesis of [11C]PBR06 and [18F]PBR06 as
agents for positron emission tomographic (PET) imaging of the translocator protein
(TSPO). Steroids. 2011;76:1331–1340.
7.35 (s, 2H, Ph−H), 7.66 (s, 2H, NH2). MS (ESI): 214 ([M+H]+, 100%); MS (ESI): 212
([M−H]−, 70%). (f). General procedure for synthesis of compounds 5a-c: Compound 4 (213
mg, 1.0 mmol) and 3-substituted benzoic acid (1.0 mmol) were dissolved in 10 mL of dry
N,N-dimethylformamide (DMF), HBTU (417 mg, 1.1 mmol) and DIPEA (258 mg,
2.0 mmol) were added, and the reaction mixture was stirred at RT for 18 h. Then the
reaction mixture was concentrated in vacuo. The residue was washed with water, and
dried in air to give crude product, which was purified by column chromatography on
silica gel with eluent (1:99 to 5:95 MeOH/CH2Cl2) to afford a white solid 5. Methyl 3-
((2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate
(5a): Yield 63%, Rf = 0.52 (1:13 MeOH/CH2Cl2), mp 252–254 °C. 1H NMR (acetone-d6):
3.90 (s, 3H, OCH3), 7.35 (s, 2H, Ph−H), 7.71 (t, J = 8.0 Hz, 1H, Ph−H), 8.23 (dt,
J = 1.5, 8.0 Hz, 1H, Ph−H), 8.39 (dt, J = 1.5, 8.0 Hz, 1H, Ph−H), 8.72 (t, J = 1.5 Hz,
1H, Ph−H). MS (ESI): 376 ([M+H]+, 25%); MS (ESI): 374 ([M−H]−, 100%). 3-(Ben-
zyloxy)-N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)benzamide
(5b): Yield 18%, Rf = 0.67 (1:9 MeOH/CH2Cl2), mp 221–223 °C. 1H NMR (acetone-d6):
7.30–7.32 (m, 3H, Ph−H), 7.33 (dt, J = 2.0, 7.0 Hz, 1H, Ph−H), 7.39 (tt, J = 1.5, 8.5 Hz,
2H, Ph−H), 7.45–7.51 (m, 3H, Ph−H), 7.76 (td, J = 1.0, 8.0 Hz, 1H, Ph−H), 7.80 (t,
J = 2.0 Hz, 1H, Ph−H), 11.80 (br s, 1H, NH). MS (ESI): 424 ([M+H]+, 9%); MS (ESI):
422 ([M−H]−, 100%). N-(2,2-Difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3-methoxybenzamide (5c): Yield 21%, Rf = 0.54 (1:13 MeOH/CH2Cl2), mp 241–243 °C.
1H NMR (DMSO-d6): 3.87 (s, 3H, OCH3), 7.21 (ddd, J = 1.0, 2.5, 8.0 Hz, 1H, Ph−H),
7.31–7.33 (br s, 2H, Ph−H), 7.47 (t, J = 8.0 Hz, 1H, Ph−H), 7.70 (t, J = 7.0 Hz, 1H,
Ph−H), 7.73 (dd, J = 1.0, 1.5 Hz, 1H, Ph−H), 7.75 (td, J = 1.0, 1.5 Hz, 1H, Ph−H),
11.80 (br s, 1H, NH). MS (ESI): 348 ([M+H]+, 25%); MS (ESI): 346 ([M−H]−, 100%).
(g). 3-((2,2-Difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic
28. Wang M, Gao M, Miller KD, Sledge GW, Zheng Q-H. [11C]GSK2126458 and
[
18F]GSK2126458, the first radiosynthesis of new potential PET agents for imaging of
PI3K and mTOR in cancers. Bioorg Med Chem Lett. 2012;22:1569–1574.
29. Wang M, Gao M, Zheng Q-H. Fully automated synthesis of PET TSPO radioligands
[
11C]DAA1106 and [18F]FEDAA1106. Appl Radiat Isot. 2012;70:965–973.
30. Mock BH, Zheng Q-H, DeGrado TR. A multi-purpose 11C-radio-synthesis system. J
Labelled Compd Radiopharm. 2005;48:S225.
31. Mock BH, Glick-Wilson BE, Zheng Q-H, DeGrado TR. Automated measurement of
specific activity of radiolabeled ligands during synthesis. J Labelled Compd
Radiopharm. 2005;48:S224.
32. Allard M, Fouquet E, James D, Szlosek-Pinaud M. State of art in 11C labelled radio-
tracers synthesis. Curr Med Chem. 2008;15:235–277.
33. Zheng Q-H, Glick-Wilson BE, Steele B, Shaffer M, Corbin L, Green MA. A simplified
conventional manual C18 Light Sep-Pak system for purification and reformulation of
carbon-11 PET tracers. J Labelled Compd Radiopharm. 2015;58:S392.
34. Wang M, Gao M, Xu Z, Zheng Q-H. Synthesis of a PET tau tracer [11C]PBB3 for
imaging of Alzheimer’s disease. Bioorg Med Chem Lett. 2015;25:4587–4592.
35. Gao M, Wang M, Green MA, Hutchins GD, Zheng Q-H. Synthesis of
[
11C]GSK1482160 as a new PET agent for targeting P2X7 receptor. Bioorg Med Chem
Lett. 2015;25:1965–1970.
36. Gao M, Wang M, Zheng Q-H. Synthesis of [11C]MK-1064 as a new PET radioligand
for imaging of orexin-2 receptor. Bioorg Med Chem Lett. 2016;26:3694–3699.
37. Zheng Q-H, Mock BH. Purification of carbon-11 PET radiotracers from unlabeled
precursors by preparative HPLC and SPE. Biomed Chromatogr. 2005;19:671–676.
4

M. Gao et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
acid (6a): Potassium hydroxide (KOH, 0.5 g, 7.6 mmol) was added into the solution of
compound 5a (375 mg, 1.0 mmol) in methanol (20 mL). The reaction mixture was stirred
at RT for 15 h. Then the reaction mixture was concentrated in vacuo, and HCl (1 N) was
added to adjust pH to 7. The resulting precipitate was filtered, washed with cold water,
dried in air to give a white solid 6a (336 mg, 93%), Rf = 0.20 (1:9 MeOH/CH2Cl2),
mp > 310 °C. 1H NMR (DMSO-d6): 7.48 (s, 2H, Ph−H), 7.63 (t, J = 7.5 Hz, 1H, Ph−H),
8.16 (d, J = 7.5 Hz, 1H, Ph−H), 8.26 (d, J =7.5 Hz, 1H, Ph−H), 12.55 (br s, 1H, OH).
MS (ESI): 362 ([M+H]+, 15%); MS (ESI): 360 ([M−H]−, 25%). HRMS (ESI): calcd for
(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 58 μA beam current
and 20 min on target. The production run produced approximately 37.0 GBq of [11C]CO2
at EOB. The precursor 6a or 6b (0.1–0.3 mg) was dissolved in CH3CN (300 μL). To this
solution was added aqueous NaOH (2 N, 2 μL). The mixture was transferred to a small
reaction vial. No-carrier-added (high molar activity) [11C]CH3OTf that was produced by
the gas-phase production method26 within 12 min from [11C]CO2 through [11C]CH4 and
C
16H10N3O5F2, 362.0589 ([M+H]+), found 362.0579. (h). N-(2,2-Difluoro-5H-[1,3]di-
[
11C]CH3Br with AgOTf column was passed into the reaction vial at RT until radioactivity
oxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b): BF3·OEt2 (0.8 mL) and
Me2S (1.0 mL) were added to a solution of compound 5b (212 mg, 0.5 mmol) in di-
chloromethane (20 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h and at RT
for 2 h. Then the reaction mixture was evaporated to dryness in vacuo. The residue was
suspended in a mixture of aqueous NaHCO3, and extracted with EtOAc (3 × 80 mL). The
combined organic layers were washed with brine, dried over Na2SO4, and concentrated.
The residue was purified by column chromatography on silica gel with eluent (2:98
MeOH/CH2Cl2) to give a white solid 6b (109 mg, 65%), Rf = 0.50 (7:93 MeOH/CH2Cl2),
mp 249–251 °C. 1H NMR (acetone-d6): 7.12 (ddd, J = 0.5, 2.5, 8.5 Hz, 1H, Ph−H), 7.30
(br s, 2H, Ph−H), 7.38 (t, J = 7.0 Hz, 1H, Ph−H), 7.65 (ddd, J = 0.5, 2.5, 8.5 Hz, 1H,
Ph−H), 11.77 (br s, 1H, OH). 13C NMR (acetone-d6): 113.44, 115.54, 115.79, 118.49,
119.94, 120.55, 130.76, 135.39, 140.32, 143.37, 148.67, 158.61, 167.45. MS (ESI): 334
([M+H]+, 60%); MS (ESI): 332 ([M−H]−, 100%). HRMS (ESI): calcd for C15H10N3O4F2,
334.0639 ([M+H]+), found 334.0625. (i) [11C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([11C]5a) and N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-[11C]methoxybenzamide ([11C]5c):
reached a maximum (2 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 aqueous NaHCO3 (0.1 M,
1 mL). The reaction vial was connected to a 3-mL HPLC injection loop. The labeled
product mixture solution was injected onto the semi-preparative HPLC column for pur-
ification. The product [11C]5a or [11C]5c fraction was collected in a recovery vial con-
taining 30 mL water. The diluted tracer solution was then passed through a C-18 Light
Sep-Pak cartridge, and washed with water (3 × 10 mL). The cartridge was eluted with
EtOH (3 × 0.4 mL) to release the labeled product, followed by saline (10–11 mL). The
eluted product was then sterile-filtered through a Millex-FG 0.2 μm membrane into a
sterile vial. Total radioactivity was assayed and total volume (10–11 mL) was noted for
tracer dose dispensing. The overall synthesis time including HPLC-SPE purification and
reformulation was ∼40 min from EOB. The decay corrected radiochemical yield was
40–45%. Retention times in the analytical HPLC system were: tR 6a = 4.77 min, tR
5a = 6.13 min, tR
5c = 6.41 min. Retention times in the preparative HPLC system were: tR 6a = 5.85 min,
tR 5a = 10.02 min, tR
11C]5a = 10.18 min; and tR 6b = 6.05 min, tR 5c = 10.23 min, tR
11C]5c = 10.38 min.
[ [
11C]5a = 6.21 min; and tR 6b = 4.82 min, tR 5c = 6.34 min, tR 11C]
[
[
11C]CO2 was produced by the 14N(p,α)11C nuclear reaction in the small volume
[
5