Synthesis of [11C]HG-10-102-01 as a new potential PET agent for imaging of LRRK2 enzyme in Parkinson's disease

Article abstract of DOI:10.1016/j.bmcl.2017.02.019

The reference standard (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(morpholino)methanone (HG-10-102-01) and its precursor (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-hydroxyphenyl)(morpholino)methanone (desmethyl-HG-10-102-

Full text of DOI:10.1016/j.bmcl.2017.02.019

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of [11C]HG-10-102-01 as a new potential PET agent
for imaging of LRRK2 enzyme in Parkinson’s disease
Min Wang 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 (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(mor-
pholino)methanone (HG-10-102-01) and its precursor (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)
amino)-3-hydroxyphenyl)(morpholino)methanone (desmethyl-HG-10-102-01) were synthesized from
2,4,5-trichloropyrimide and 3-methoxy-4-nitrobenzoic acid with overall chemical yield 49% in four steps
and 14% in five steps, respectively. The target tracer (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)
amino)-3-[11C]methoxyphenyl)(morpholino)methanone ([11C]HG-10-102-01) was prepared from the
precursor desmethyl-HG-10-102-01 with [11C]CH3OTf through O-[11C]methylation and isolated by
HPLC combined with SPE in 45–55% radiochemical yield, based on [11C]CO2 and decay corrected to
end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB
Received 25 January 2017
Revised 7 February 2017
Accepted 8 February 2017
Available online xxxx
Keywords:
[
11C]HG-10-102-01
Leucine-rich repeat kinase 2 (LRRK2)
Radiosynthesis
Positron emission tomography (PET)
Parkinson’s disease (PD)
was 370–1110 GBq/
lmol with a total synthesis time of ꢀ40-min from EOB.
Ó 2017 Elsevier Ltd. All rights reserved.
Parkinson’s disease (PD) is the second most common neurode-
generative disease of the central nervous system (CNS) that mainly
affects the motor system in elderly people.1–3 The exact cause is
still unknown, and thus there is presently no cure. Current stan-
dards of care have serious limitations and largely address only
symptoms, and the major treatment options are medication and
surgery to manage PD’s symptoms.4–6 To discover more effective
treatments, a reliable diagnostic tool is really needed.7 Neuroimag-
ing of PD is one of the most active as well as most challenging areas
in neuroscience.8,9 Advanced biomedical imaging technique posi-
tron emission tomography (PET) is a promising modality for PD,
and significant advances have accomplished in this field of molec-
ular imaging.10 The representative PET tracers used in PD imaging
such as [18F]FDOPA for dopamine synthesis, [11C]b-CFT for dopa-
mine transporter, [11C]DTBZ for vesicular monoamine transporter
type 2, [11C]raclopride for D2 receptor, [11C]WAY100635 for sero-
tonin receptor, [11C]PBR28 for translocator protein, [11C]PIB for
aggregated b-amyloid plaques, and [18F]T807 ([18F]AV-1451) for
tau protein are listed in Fig. 1.11–16
therapies, as currently only symptomatic treatment is
available.17–19 Recent genetic studies provide new opportunity to
discover molecularly targeted therapeutics for neurodegenera-
tion.20,21 Among the genes associated with PD, leucine-rich repeat
kinase 2 (LRRK2) is a unique enzyme in humans linked to an
increased risk of PD, the most common known cause of parkinson-
ism.22 LRRK2 has become an attractive therapeutic target for PD,
many highly potent, selective, and brain-penetrable LRRK2 small
molecule inhibitors have been developed, and the lead compounds
HG-10-102-01
((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)
amino)-3-methoxyphenyl)(morpholino)methanone), G1023 and
G7915 exhibited excellent biochemical IC50 values of 3.2 nM
against LRRK2[G2019S], 9 nM against pLRRK2, and 9 nM against
pLRRK2, respectively.4,5,23–27 LRRK2 has also become a promising
imaging target for PD, since the efforts in developing therapeutic
agents for LRRK2 enzyme have been accompanied by a growing
interest in translating therapeutic agents to diagnostic agents.
We are interested in the development of new PET agents for PD
imaging. In our previous works, we have synthesized various PET
PD imaging agents like [11C]b-CFT, [11C]b-CIT, [11C]raclopride,
The development of new PET imaging probes for in vivo
detection of PD is critical for early and accurate diagnosis and for
the successful discovery of disease-modifying or neuro-protective
[
18F]fallypride, [11C]PBR28, [11C]PIB and [18F]T807.28–34 In this
ongoing effort, we target LRRK2 and develop radiolabeled LRRK2
inhibitors as new potential PET agents for imaging of LRRK2
enzyme in PD.
The PubMed search showed no records on radiolabeled LRRK2
inhibitors. The Google search indicated that Genentech has filed
⇑
Corresponding author.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
http://dx.doi.org/10.1016/j.bmcl.2017.02.019
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.019

2
M. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. PET tracers for imaging of PD.
a patent entitled fluorine-18 and carbon-11 labeled radioligands
for positron emission tomography (PET) imaging for LRRK2, and
several radiolabeled LRRK2 inhibitors such as 11C-G1023, 18FE-
G1023, 18FM-G1023, 11C-G7915, 18FE-G7915, and 18FM-G7915
have been patented (Fig. 2).35 However, the patented synthesis of
radiolabeled LRRK2 inhibitors is complicated or lacks synthetic
details, and certain key steps gave poor yield or were difficult to
reproduce in our hands. On the other hand, HG-10-102-01 was
reported earlier than Genentech compounds G1023 and G7915
with simpler chemistry and better binding affinity,4,5,23–27
although they have similar chemical structures. Here we report
the synthesis of a new radiolabeled LRRK2 inhibitor [11C]HG-10-
102-01 ((4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-
102-01, 5) were synthesized as depicted in Scheme 1.4,5 Commer-
cially available 2,4,5-trichloropyrimide was regioselectively
aminated with methylamine in THF to obtain 2,5-dichloro-N-
methylpyrimidin-4-amine (1) in 98% yield. Amide derivative 2
was achieved by chlorination of 3-methoxy-4-nitrobenzoic acid
with thionly chloride in toluene, followed by amination with mor-
pholine in the presence of N,N-diisopropylethylamine (DIPEA) in
THF in 75% yield. The nitro group of compound 2 was reduced by
hydrogenation under H2 with 10% Pd/C as catalyst in THF and
MeOH to afford aniline 3 in 92% yield. Amination of compound 1
by trifluoroacetic acid (TFA) catalyzed SNAr reaction of aniline 3
in 2-butanol gave reference standard 4 in 73% yield. The methoxyl
group of compound 4 was demethylated with BBr3 in CH2Cl2 to
afford the precursor desmethyl-HG-10-102-01 (5) in 28% yield.
Synthesis of [11C]HG-10-102-01 ([11C]4) is shown in Scheme 2.
Desmethyl-HG-10-102-01 underwent O-[11C]methylation36,37
using the reactive [11C]methylating agent [11C]methyl triflate
[
11C]methoxyphenyl)(morpholino)methanone).
The reference standard HG-10-102-01 (4) and its demethylated
precursor (4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-
3-hydroxyphenyl)(morpholino)methanone (desmethyl-HG-10-
Fig. 2. Fluorine-18 and carbon-11 labeled radioligands for LRRK2.
Please cite this article in press as: Wang M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.019

M. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Cl
Cl
Cl
outlet of the HPLC-portion of the system. For the reported synthe-
N
methylamine, THF
N
ses, product SA was in a range of 370–1110 GBq/lmol at EOB. The
Cl
N
Cl
N
NHCH3
major factors including [11C]-target and [11C]-radiosynthesis unit
that affect the EOB SA significantly to lead to such a wide range
1
NH2
NO2
NO2
from 370 to 1110 GBq/lmol have been discussed in our previous
H3CO
H3CO
H3CO
works.47 The general methods to increase SA have been described
as well, and the SA of our [11C]-tracers is significantly improved.47
The ‘wide range’ of SA we reported is for the same [11C]-tracer pro-
duced in different days, because very different [11C]-target and
H
2, 10% Pd/C
i) SO2Cl2, toluene
ii)morpholine, DIPEA, THF
THF/MeOH
1
, TFA, 2-butanol
O
O
N
N
N
O
O
OH
O
3
2
[
11C]-radiosynthesis unit situations would make SA in a wide
Cl
Cl
N
range. For a [11C]-tracer produced in the same day, the SA of the
same tracer in different production runs will be in a small range,
because [11C]-target and [11C]-radiosynthesis unit would not be
much different in the same day. Likewise, the methods to minimize
such wide range of SA from practice perspective have been pro-
vided in our previous works.47 At the end of synthesis (EOS), the
SA of [11C]-tracer was determined again by analytical HPLC,48
calculated, decay corrected to EOB, and based on [11C]CO2, which
was in agreement with the ‘on line’ determined value. In each
our [11C]-tracer production, if semi-preparative HPLC was used
for purification, then the SA of [11C]-tracer was assessed by both
semi-preparative HPLC (during synthesis) and analytical HPLC
(EOS); if SPE was used for purification, then the SA of [11C]-tracer
was only measured by analytical HPLC at EOS.37
N
NHCH3
HN
N
NHCH3
BBr3, CH2Cl2
HN
HO
H3CO
O
N
O
N
O
O
5
4
Scheme 1. Synthesis of HG-10-102-01 (4) and desmethyl-HG-10-102-01 (5).
([11C]CH3OTf)38,39 in acetonitrile at 80 °C under basic conditions
(2 N NaOH). The product was isolated by semi-preparative
reverse-phase (RP) high performance liquid chromatography
(HPLC) with a C-18 column, and then concentrated by solid-phase
extraction (SPE)40,41 with a disposable C-18 Light Sep-Pak cartridge
to produce the corresponding pure radiolabeled compound [11C]4
in 45–55% radiochemical yield, decay corrected to end of bombard-
ment (EOB), based on [11C]CO2.
Chemical purity and radiochemical purity were determined by
analytical HPLC.48 The chemical purity of the precursor and refer-
ence standard was >90%. The radiochemical purity of the target
tracer was >99% determined by radio-HPLC through
c-ray (PIN
The radiosynthesis included three stages: (1) labeling reaction;
(2) purification; and (3) formulation. We employed more reactive
diode) flow detector, and the chemical purity of the target tracer
was >90% determined by reversed-phase HPLC through UV flow
detector.
[
11C]CH3OTf, instead of commonly used [11C]methyl iodide ([11C]
CH3I),42 in O-[11C]methylation to improve radiochemical yield of
11C]4. We used an Eckert & Ziegler Modular Lab C-11 Methyl
Iodide/Triflate module to produce [11C]methylating agent either
11C]CH3OTf or [11C]CH3I ([11C]CH3Br passed through a NaI col-
ChemOffice 2015 indicated that the LogP for desmethyl-HG-10-
102-01 and HG-10-102-01 is 1.43 and 1.7, respectively. We assume
the retention time of HG-10-102-01 should be longer than the
retention time of desmethyl-HG-10-102-01 in RP-HPLC. However,
the precursor has longer retention time than reference standard.
This atypical elution order is difficult to explain, but fortunate from
the radiosynthetic standpoint, because the radiochemical product
eluted before the precursor. We experienced the same case when
we radiosynthesized [11C]b-CIT from Nor-b-CIT precursor.29 It is
important to note that the LogP for Nor-b-CIT and b-CIT is 3.31
and 3.69, respectively, from ChemOffice 2015.
[
[
umn). The direct comparison between [11C]CH3OTf and [11C]CH3I
confirmed the result. The labeling reaction was conducted using
a V-vial method. Addition of aqueous NaHCO3 to quench the radi-
olabeling reaction and to dilute the radiolabeling mixture prior to
the injection onto the semi-preparative HPLC column for purifica-
tion gave better separation of [11C]4 from its phenyl hydroxyl pre-
cursor 5. We used Sep-Pak trap/release method instead of rotatory
evaporation for formulation to improve the chemical purity of
radiolabeled product [11C]4. In addition, a C18 Light Sep-Pak to
replace a C18 Plus Sep-Pak allowed final product formulation with
ꢁ5% ethanol.43 Overall, it took ꢀ40 min for synthesis, purification
and dose formulation.
The experimental details and characterization data for com-
pounds 1–5 and for the tracer [11C]4 are given.49
In summary, synthetic routes with moderate to high yields have
been developed to produce HG-10-102-01, desmethyl-HG-10-102-
01 and [11C]HG-10-102-01. The radiosynthesis employed [11C]CH3-
OTf for O-[11C]methylation at the phenyl hydroxyl position of the
desmethyl precursor, followed by product purification and isola-
tion using a semi-preparative RP HPLC combined with SPE. [11C]
HG-10-102-01 was obtained in high radiochemical yield, radio-
chemical purity and chemical purity, with a reasonably short over-
all synthesis time, and high specific activity. This will facilitate
studies to evaluate [11C]HG-10-102-01 as a new potential PET
agent for imaging of LRRK2 enzyme in PD.
The radiosynthesis was performed in a home-built automated
multi-purpose [11C]-radiosynthesis module.44–46 This radiosynthe-
sis module facilitated the overall design of the reaction, purifica-
tion and reformulation capabilities in a fashion suitable for
adaptation to preparation of human doses. In addition, the module
is designed to allow in-process measurement of [11C]-tracer speci-
fic activity (SA, GBq/
l
mol at EOB) using a radiation detector at the
Cl
Cl
N
N
Acknowledgments
N
NHCH3
HN
N
NHCH3
HN
11C]CH3OTf, CH3CN
2 N NaoH, 80 oC, 3 min
H311CO
[
HO
This work was partially supported by Indiana State Department
of Health (ISDH) Indiana Spinal Cord & Brain Injury Fund (ISDH
EDS-A70-2-079612) in the United States. 1H NMR and 13C NMR
spectra were recorded at 500 and 125 MHz, respectively, on a Bru-
ker 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
O
N
O
O
N
O
11
5
4
C]
[
Scheme 2. Synthesis of [11C]HG-10-102-01 ([11C]4).
Please cite this article in press as: Wang M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.019

4
M. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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 (Phenom-
States National Science Foundation (NSF) Major Research Instru-
mentation Program (MRI) grant CHE-0619254.
enex) 5
60% H2O to 70%CH3CN/30%H2O within 10 min; flow rate 1.0 mL/min; UV
(254 nm) and -ray (PIN diode) flow detectors. Semi-preparative RP HPLC was
performed using a Prodigy (Phenomenex) 5
m C-18 column, 10 Â 250 mm;
mobile phase 43%CH3CN/57%H2O; flow rate 7 mL/min; UV (254 nm) and -ray
(PIN diode) flow detectors. C18 Light Sep-Pak cartridges were obtained from
Waters Corporation (Milford, MA). Sterile Millex-FG 0.2 m filter units were
obtained from Millipore Corporation (Bedford, MA).(b). 2,5-Dichloro-N-
methylpyrimidin-4-amine (1): To a stirred solution of 2,4,5-trichloropyrimide
(3.0 g, 16.4 mmol) in anhydrous THF (45 mL) was added methylamine (2.0 M
solution in THF, 25 mL, 50.0 mmol) dropwise at 0 °C. After the reaction mixture
was warmed to room temperature (RT) and stirred overnight, it was diluted
with water and extracted with EtOAc. The combined organic layer was washed
with brine, dried over anhydrous Na2SO4 and filtered. The solvent was
evaporated in vacuo to afford 1 as a white solid (2.9 g, 98%), which was used
for next step without purification. 1H NMR (DMSO-d6): d 8.14 (s, 1H), 7.91 (br s,
1H), 2.87 (d, J = 3.0 Hz, 3H).(c). (3-Methoxy-4-nitrophenyl)(morpholino)metha-
lm C-18 column, 4.6 Â 250 mm; a gradient mobile phase 40%CH3CN/
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25.4 mmol) in anhydrous toluene (50 mL) was added thionyl chloride (6 mL,
82.3 mmol). The reaction mixture was heated under reflux for 2 h, and the
solvent was evaporated in vacuo. To the residual was added anhydrous THF
(50 mL), followed by DIPEA (9.1 mL, 50.7 mmol) and morpholine (3.3 mL,
38.0 mmol) at 0 °C. After the reaction mixture was warmed to RT and stirred for
1 h, it was diluted with water and extracted with EtOAc. The combined organic
layer was washed with brine, dried over anhydrous Na2SO4 and filtered. The
solvent was evaporated in vacuo. The crude product was purified by silica gel
column chromatography with CH2Cl2/MeOH (100:1 to 100:3) as eluent to
afford 2 as a yellow solid (5.04 g, 75%); mp 104–105 °C. 1H NMR (DMSO-d6): d
7.93 (d, J = 8.5 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.12 (dd, J = 1.5, 8.0 Hz, 1H), 3.95
(s, 3H), 3.67 (m, 2H), 3.63 (m, 2H), 3.55 (m, 2H), 3.30 (m, 2H).(d). (4-Amino-3-
methoxyphenyl)(morpholino)methanone (3): A solution of compound 2 (5.0 g,
18.8 mmol) in anhydrous THF (15 mL) and MeOH (50 mL) was hydrogenated
with H2 (60 psi) over 10% Pd-C (520 mg) at RT for 5 h. The catalyst was
removed by filtration, and the solvent was evaporated in vacuo. The crude
product was purified by silica gel column chromatography with CH2Cl2/MeOH
(100:1–100:8) as eluent to afford 2 as a white solid (4.09 g, 92%); mp 146–
148 °C. 1H NMR (DMSO-d6): d 6.85 (d, J = 1.5 Hz, 1H), 6.80 (dd, J = 2.0, 8.0 Hz,
1H), 6.61 (d, J = 8.0 Hz, 1H), 5.15 (s, 2H), 3.77 (s, 3H), 3.59–3.57 (m, 4H), 3.51–
3.49 (m, 4H).(e). (4-((5-Chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-meth-
oxyphenyl)(morpholino)methanone (HG-10-102-01, 4): To a stirred suspension
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of compound
1 (300 mg, 1.68 mmol) in 2-butanol (3 mL) was added TFA
(0.3 mL), followed by the addition of compound 3 (380 mg, 1.61 mmol). The
reaction mixture was heated under reflux overnight, and the solvent was
evaporated in vacuo. The residual was diluted with CH2Cl2, washed with
saturated aqueous NaHCO3 and brine. The organic layer was dried over
anhydrous Na2SO4 and filtered. The solvent was evaporated in vacuo. The crude
product was purified by silica gel column chromatography with CH2Cl2/MeOH
(100:5) as eluent to afford 4 as a white solid (350 mg, 73%); mp 167–169 °C. 1
H
NMR (DMSO-d6): d 8.41 (d, J = 8.5 Hz, 1H), 7.96 (s, 1H), 7.68 (s, 1H), 7.33–7.30
(m, 1H), 7.05 (d, J = 1.5 Hz, 1H), 7.01 (dd, J = 1.5, 8.0 Hz, 1H), 3.90 (s, 3H), 3.60
(m, 4H), 3.52 (m, 4H), 2.91 (d, J = 4.5 Hz, 3H). 13C NMR (DMSO-d6): d 169.0,
158.1, 157.4, 152.4, 147.2, 130.5, 128.0, 119.8, 117.4, 109.8, 104.9, 66.1, 56.0,
27.7. LC-MS (ESI, m/z): Calcd for C17H21ClN5O3 ([M+H]+) 378.1, found: 378.2.
HRMS (ESI, m/z): Calcd for C17H21ClN5O3 ([M+H]+) 378.1333, found: 378.1345.
(f). (4-((5-Chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-hydroxyphenyl)(mor-
pholino)methanone (desmethyl-HG-10-102-01, 5): To
a stirred solution of
compound 4 (345 mg, 0.91 mmol) in anhydrous CH2Cl2 (6 mL) was added
BBr3 (1.0 M solution in CH2Cl2, 4 mL, 4.0 mmol) dropwise at -78 °C under N2
atmosphere. After the reaction mixture was warmed to RT and stirred
overnight, it was quenched with ice-water and neutralized with aqueous 1 N
NaHCO3 solution. The mixture was extracted with CH2Cl2. The combined
organic layer was washed with brine, dried over anhydrous Na2SO4 and
filtered. The solvent was evaporated in vacuo. The crude product was purified
by preparative TLC plates with CH2Cl2/MeOH (150:7) as eluent to afford 5 as an
off-white solid (94 mg, 28%); mp 213–215 °C. 1H NMR (DMSO-d6): d 10.4 (br s,
1H), 8.19 (d, J = 8.4 Hz, 1H), 7.95 (s, 1H), 7.86 (s, 1H), 7.34–7.31 (m, 1H), 6.91 (d,
J = 2.0 Hz, 1H), 6.87 (dd, J = 2.0, 8.4 Hz, 1H), 3.59–3.58 (m, 4H), 3.50 (m, 4H),
2.92 (d, J = 4.4 Hz, 3H). 13C NMR (DMSO-d6): d 169.6, 158.6, 158.1, 152.6, 146.1,
130.4, 128.9, 119.0, 118.8, 114.6, 105.1, 66.7, 28.2. LC-MS (ESI, m/z): Calcd for
46. Wang M, Gao M, Zheng Q-H. Appl Radiat Isot. 2012;70:965–973.
47. Gao M, Wang M, Zheng Q-H. Bioorg Med Chem Lett. 2017;27:740–743.
48. Zheng Q-H, Mock BH. Biomed Chromatogr. 2005;19:671–676.
49. (a). General: All commercial reagents and solvents were purchased from Sigma-
Aldrich and Fisher Scientific, and used without further purification.
CH3OTf was prepared according to a literature procedure.39 Melting points
were determined on MEL-TEMP II capillary tube apparatus and were
[
11C]
a
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 (d) are reported in parts per million (ppm) relative to an
internal standard tetramethylsilane (TMS, d 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
C
H
16H19ClN5O3 ([M+H]+) 364.1, found: 364.1. HRMS (ESI, m/z): Calcd for C16
19ClN5O3 ([M+H]+) 364.1176, found: 364.1182.(g). (4-((5-Chloro-4-(methy-
lamino)pyrimidin-2-yl)amino)-3-[11C]methoxyphenyl)(morpholino)methanone
-
([11C]HG-10-102-01, [11C]4): [11C]CO2 was produced by the 14N(p, 11C nuclear
a)
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
and
electrospray ionization. The high resolution mass spectra (HRMS) were
obtained using Waters/Micromass LCT Classic spectrometer. Chromato-
a 1946D mass spectrometer configured for positive-ion/negative-ion
irradiations used for the development were 58 lA beam current and 15 min on
a
target. The production run produced approximately 25.9 GBq of [11C]CO2 at
graphic 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. Preparative TLC was run
using Analtech silica gel UV254 plates (20 Â 20 cm2). Normal phase flash
column chromatography was carried out on EM Science silica gel 60 (230–
EOB. Desmethyl-HG-10-102-01 (5, 0.1–0.3 mg) was dissolved in CH3CN
(300
was transferred to
activity) [11C]CH3OTf that was produced by the gas-phase production method39
lL). To this solution was added aqueous NaOH (2 N, 2
lL). The mixture
small reaction vial. No-carrier-added (high specific
a
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M. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
within 12 min from [11C]CO2 through [11C]CH4 and [11C]CH3Br with silver
triflate (AgOTf) column was passed into the reaction vial at RT until radioac-
tivity 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 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 Light cartridge, and washed with
water (3 Â 10 mL). The cartridge was eluted with EtOH (3 Â 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 m membrane into a sterile vial.
l
Total radioactivity was assayed and total volume (10–11 mL) was noted for
tracer dose dispensing. The overall synthesis time including HPLC-SPE purifi-
cation and reformulation was ꢀ40 min from EOB. The decay corrected
radiochemical yield was 45–55%. Retention times in the analytical HPLC
system were: tR 5 = 7.87 min, tR 4 = 6.05 min, tR
times in the preparative HPLC system were: tR 5 = 17.65 min, tR 4 = 11.32 min,
tR
11C]4 = 11.58 min.
[
11C]4 = 6.16 min. Retention
[
Please cite this article in press as: Wang M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.019