Evaluation of [18F]-ATRi as PET tracer for in vivo imaging of ATR in mouse models of brain cancer

Article abstract of DOI:10.1016/j.nucmedbio.2017.01.002

Rationale Ataxia telangiectasia and Rad3-related (ATR) threonine serine kinase is one of the key elements in orchestrating the DNA damage response (DDR). As such, inhibition of ATR can amplify the effects of chemo- and radiation-therapy, and several ATR i

Full text of DOI:10.1016/j.nucmedbio.2017.01.002

Evaluation of [18F]-ATRi as PET tracer for in vivo imaging of ATR in mouse
models of brain cancer
Giuseppe Carlucci, Brandon Carney, Ahmad Sadique, Axel Vansteene,
Jun Tang, Thomas Reiner
PII:
DOI:
Reference:
S0969-8051(16)30293-1
doi: 10.1016/j.nucmedbio.2017.01.002
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To appear in:
Nuclear Medicine and Biology
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Revised date:
Accepted date:
21 September 2016
28 November 2016
5 January 2017
Please cite this article as: Carlucci Giuseppe, Carney Brandon, Sadique Ahmad,
Vansteene Axel, Tang Jun, Reiner Thomas, Evaluation of [18F]-ATRi as PET tracer for
in vivo imaging of ATR in mouse models of brain cancer, Nuclear Medicine and Biology
(2017), doi: 10.1016/j.nucmedbio.2017.01.002
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Evaluation of [18F]-ATRi as PET tracer for in
vivo imaging of ATR in mouse models of brain
cancer
a
a
a
a
Giuseppe Carlucci , Brandon Carney a, b, Ahmad Sadique , Axel Vansteene , Jun Tang ,
Thomas Reiner a, c,
*
a Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065,
USA
b Department of Chemistry, Hunter College and PhD Program in Chemistry, The Graduate
Center of the City University of New York, New York, NY 10018, USA
c Weill Cornell Medical College, New York, NY, 10065, USA
Abbreviated title: In vivo imaging of ATR in mouse models of brain cancer
Keywords: ATR, cell cycle, PET imaging, U251 MG, Ve-821, 18F
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Abstract
Rationale: Ataxia telangiectasia and Rad3-related (ATR) threonine serine kinase is one of the
key elements in orchestrating the DNA damage response (DDR). As such, inhibition of ATR can
amplify the effects of chemo- and radiation-therapy, and several ATR inhibitors (ATRi) have
already undergone clinical testing in cancer. For more accurate patient selection, monitoring and
staging, real-time in vivo imaging of ATR could be invaluable; the development of appropriate
imaging agents has remained a major challenge. Methods: 3-amino-N-(4-[18F]phenyl)-6-(4-
(methylsulfonyl)phenyl)pyrazine-2-carboxamide ([18F]-ATRi), a close analogue of Ve-821, (a
clinical ATRi candidate), was readily accomplished similarly to already established synthetic
18
procedures. Structurally, F was introduced at the 4-position of the aromatic ring of Ve-821 for
generating a labeled ATR inhibitor. In vitro experiments were conducted in U251 MG
glioblastoma cell lines and ex vivo biodistribution were performed in subcutaneous U251 MG
xenograft bearing athymic nude mice following microPET imaging. Results: [18F]-ATRi has a
similar pharmacokinetic profile to that of Ve-821. Using an U251 MG glioblastoma mouse
model, we evaluated the in vivo binding efficiency of [18F]-ATRi. Blood and tumor showed a
statistically significant difference between mice injected with only the probe or following
blocking experiment with Ve-821 (1.48 ± 0.40 %ID/g vs. 0.46 ± 0.12 %ID/g in tumor and 1.85 ±
0.47 %ID/g vs. 0.84 ± 0.3 %ID/g in blood respectively). Conclusions: [18F]-ATRi represents the
18
first F positron emission tomography (PET) ATR imaging agent, and is designed on a low
nanomolar and clinically relevant ATR inhibitor.
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Introduction
One of the most fundamental functions of cells is to maintain the integrity of their genomes [1-
5]. Sophisticated cell cycle checkpoint pathways are in place to respond and remediate DNA
damage quickly, and to efficiently control cycle progression and DNA replication [5, 6]. The
overall function of these checkpoints is to detect damaged or abnormally structured DNA, and to
coordinate cell-cycle progression with DNA repair. Fundamentally speaking, cell-cycle
checkpoints control cell-cycle phase transitions and ensure the correct completion of prior events
[7]. ATR protein kinase and ataxia telangiectasia mutated (ATM) are two central kinases of such
checkpoints pathways [7-10]. Both are activated by DNA damage and DNA replication stress,
but their DNA-damage specificities are distinct and their functions are not redundant [7, 8, 11].
The serine/threonine-protein kinase ATR is involved in the DNA damage repair (DDR)
mechanism and is activated by single stranded DNA structures, which may occur at resected
DNA double strand breaks (DSBs) or stalled replication forks [12-14]. When DNA polymerases
stall during DNA replication, replicative helicases continue to unwind the DNA ahead of the
replication fork, leading to the generation of long stretches of single stranded DNA (ssDNA),
which are then bound by the single-strand binding protein complex Replication protein A (RPA)
[7]. Once activated, ATR triggers Checkpoint-1 (Chk1) and other downstream targets to promote
DNA repair, stabilization, restart of stalled replication forks and transient cell cycle arrest [9, 15,
16]. A growing body of data, however, now shows roles for ATR far beyond the activation of
Chk1 [17-20] where ATR also plays a role in normal replication of undamaged DNA [12]. Over
the last decade, a variety of studies have also suggested that checkpoint inhibition might indeed
selectively sensitize cancer cells [17, 19, 21-26], and many current cancer treatments, including
chemotherapeutic agents and ionizing radiation, induce DNA damage and replication fork
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stalling, thereby activating cell cycle checkpoint pathways [6, 11, 17]. Following this, efficient
inhibition of ATR would enhance and amplify the chemotherapeutic effects of such treatments,
and variety of studies have indeed shown that selective inhibition of ATR and Chk1 enhances the
killing of tumor cells [17, 19, 25, 27]. The ATR/Chk1 cell cycle pathway is often upregulated,
and loss or mutations in human cancers is low [8, 9, 11, 25]. Therefore, ATR could be ideal
target for PET imaging agent development. Non-invasive imaging of ATR is of high clinical
relevance, not only for patient selection, but potentially also for measuring target engagement
during combination therapies. With this goal in mind, we set out to develop [18F]-ATRi, a PET
radiolabeled version of the ATR inhibitor Ve-821, which non-invasively images kinase
expression in animal model of cancer (Fig. 1). Specifically, our goal was to determine if: i) [18F]-
ATRi has suitable pharmacokinetic properties for imaging ATR, and ii) whether the tracer is
selective. Herein, we report on the chemical synthesis, radiolabeling and preclinical evaluation of
this new ATR based Ve-821 imaging agent. For both in vitro as well as in vivo evaluation we
used U251 MG cells, a human glioblastoma cell line. We show that our imaging agent could be a
valuable probe targeting ATR for tumor imaging and also demonstrating that ATR activation as
a biological process could have a strong prognostic value in the future.
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Material and Methods
Reagents and instrumentation
Reagents were purchased from Acros and Sigma-Aldrich Co. and used without further
purification unless otherwise noted. Ve-821 was obtained from Aldrich (Sigma-Aldrich Co., St.
13
Louis, MO). Proton and Carbon nuclear magnetic resonance (1H and C NMR) spectra were
recorded on a Bruker Daltonics (600 MHz) spectrometer (Bruker, Billerica, MA). Chemical shift
of protons and carbons were analyzed against the DMSO lock signal and reported as parts per
million (ppm). Phosphate buffered saline (PBS) and Dulbecco’s Modified Eagle Medium
(DMEM) was purchased from the media preparation facility at Memorial Sloan Kettering Cancer
Center (New York, NY, USA). U251 MG, a human glioblastoma cancer cell line was purchased
from ATCC (Manassas, VA). Semi-preparative high performance liquid chromatography
(HPLC) purification was achieved using a Luna C18 250 mm x 10 mm (Phenomenex, Torrance,
CA) (Method A : eluents A = 0.1% TFA in water and B = 0.1% TFA in MeCN; gradient 0–17
min, 5–80% B; 17–21 min, 80–95% B; 21–24 min, 95% B; 24–25 min, 95–5% B; 25–30 min,
5% B; 3 mL min-1). Analytical HPLC analyses were performed using a Gemini C18 250 mm x
4.6 mm reversed-phase column (Phenomenex, Torrance, CA) (Method B: eluents A = 0.1% TFA
in water and B = 0.1% TFA in MeCN; gradient 0–17 min, 5–95% B; 17–21 min, 95% B; 21–22
min, 95–5% B; 22–25 min, 5% B; 1 mL min-1). Both semi-preparative and analytical HPLC
purifications were performed on a Shimadzu UFLC HPLC system (Shimadzu Scientific
Instruments, Inc, Columbia, MD) equipped with a DGU-20A degasser, a SPD-M20A UV
detector, a LC-20AB pump system, and a Scan-RAM radio-TLC/HPLC-detector from LabLogic
(Brandon, FL) with a dual-wavelength UV–Vis detector followed by a flow-through γ detector
18
connected in series. Semi-preparative and analytical HPLC analyses of F-labeled compounds
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were calibrated with the corresponding 19F analogues. Microwave syntheses were conducted
with a Biotage® Initiator+ synthesizer (Charlotte, NC). Radioactivity in blood half-life, cell
uptake, competitive displacement and biodistribution studies was quantified with a WIZARD2
automatic γ-counter from Perkin Elmer (Boston, MA). Small animal PET imaging data were
recorded on a Focus 120 MicroPET (Concorde Microsystems, Knoxville, TN) and reconstructed
using ASI Pro VMTM MicroPET Analysis software (Siemens Medical Solutions, Knoxville, TX).
[18F]-ATRi, 19F-ATRi and Ve-821 were formulated in 20% PEG300 / 80% PBS and used
without any further filtration unless otherwise described.
Preparation of 3-amino-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carboxylic acid (3)
To a reaction tube containing 3-amino-6-bromo-N-phenyl-pyrazine-2-carboxamide 1a (88 mg,
0.3 mmol) in DMF (4 mL), 2-(4-methanesulfonylphenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane 1b (0.3 mmol) was added, followed by Pd(PPh3)2Cl2 (5 mol %) and 2 M aqueous
Na2CO3 solution (2 mL). The tubes were then flushed with N2 and heated to 110 °C for 1 h in a
microwave. After this time, the reaction was allowed to cool to ambient temperature, filtered,
and purified by reverse phase HPLC (method A) to give the desired product as solid after
lyophilization (80 ± 10 % yield). The lyophilized product methyl 3-amino-6-(4-
(methylsulfonyl)phenyl)pyrazine-2-carboxylate 2 (50 mg, 0.15 mmol) was further dissolved in
MeOH/H2O (3 mL) and reacted overnight in the presence of LiOH (11 mg, 0.45 mmol) under
reflux conditions to yield 3-amino-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carboxylic acid 3 (65
± 7 % yield). The reaction was allowed to cool to ambient temperature, filtered, and purified by
reverse phase HPLC to give the desired products as solids after lyophilization (75 ± 10 % yield).
1
+
-
HRMS(+) m/z = 294.0178 [M + H] , 292.0267 [M - H] ; H-NMR (600 MHz, DMSO-d6) δ =
9.02 (s, 1H), 8.35 (d, 3JHH = 8.5 Hz, 2H), 7.99 d, 3JHH = 8.5 Hz, 2H), 7.70 (m, 2H), 3.25 (s, 3H).
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Preparation
of
3-amino-N-(4-fluorophenyl)-6-(4-(methylsulfonyl)phenyl)pyrazine-2-
carboxamide (5)
To a solution of 3-amino-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carboxylic acid in DMSO (6
mg, 0.04 mmol), 4-fluoroaniline (0.2 mmol), HBTU (0.12 mmol) and triethylamine (0.12 mmol)
were added and the solution reacted at 150 °C for 10 min. The resulting solution was filtered and
purified by reverse phase HPLC (method A) to the desired amide derivative (yields ranged from
65% to 74%), dried and lyophilized to yield the final product 5. HRMS(+) m/z = 308.0887 [M +
1
+
-
H] , 306.0267 [M - H] ; H-NMR (600 MHz, DMSO-d6) δ = 10.52 (s, 1H), 9.03 (s, 1H), 8.50 (d,
3JHH = 8.6 Hz, 2H), 7.99 (d, 3JHH = 8.6 Hz, 2H), 7.86 (m, 2H), 7.82 (dd, 3JHH = 9.1 Hz, 3JHF = 5.0
Hz, 2H), 7.25 (t, 3JHH = 8.9 Hz, 2H), 3.26 (s, 3H).
Preparation of 3-amino-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carbonyl chloride (8)
Thionyl chloride (1 μL, 50 μmol) was added to a solution of 3-amino-6-(4-
(methylsulfonyl)phenyl)pyrazine-2-carboxylic acid in toluene (5 mg in 500 μL), and the solution
reacted at 100℃ for 3 h. The final 3-amino-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carbonyl
chloride product (80 ± 15 % yield) was stored at -80 °C until further use.
Radiochemistry
4-[18F]fluoroaniline was obtained analogously to what was reported before from Hendricks and
coworkers [28]. Briefly, a QMA cartridge containing cyclotron-produced [18F] fluoride ion was
eluted with a solution containing 9 mg Kryptofix [2.2.2] (4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane), 0.08 mL 0.15 M K2CO3 and 1.92 mL MeCN into a 5 mL
reaction vial. Water was removed azeotropically at 120 °C. 3 mg of 1,4-dinitrobenzene were
dissolved in 300 μL of DMSO, transferred to a sealed reaction vial and finally heated to 120 °C
in a microwave for 5 min and then cooled to room temperature. The reaction mixture was diluted
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with water (8 mL) and loaded onto a Sep-Pak® Light C18 (Waters, Milford, MA)
preconditioned with EtOH (10 mL) and water (10 mL). The cartridge was washed with 5 mL
MilliQ water and 4-[18F]fluoronitrobenzene (6) was eluted from the cartridge with MeOH (1
mL). To (6), 3 mg of Pd/C and 28 mg NaBH4 were added and the reaction mixture stirred for 5
min, at which time unreacted NaBH4 was quenched by adding 300 μL 6 N HCl. The mixture was
diluted with 1 N NaOH(aq) and passed through a Lichrolut EN cartridge (Merck, Billerica, MA)
and a Na2SO4: Celite (3:2 w/w). The loaded 4-[18F]fluoroaniline (7) was finally eluted with THF
(1mL) in a vial containing 500 μg of 3-amino-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carbonyl
chloride (8) and the mixture was stirred at room temperature for 5 min. Thereafter, the reaction
mixture was purified by semi-preparative HPLC (method A) yielding 28.7 ± 7% dcRCY in 120 ±
15 min and a 98 ± 2 % radiochemical purity over 3 steps preparation. The specific activity was
determined to be 0.7 Ci/μmol.
Cell culture
U251 MG cells were grown in Eagle’s Minimal Essential Medium (MEM) containing 10%
(vol/vol) heat inactivated fetal bovine serum, 100 IU penicillin, and 100 μg/mL streptomycin as
purchased from the culture media preparation facility at MSK (New York, NY).
Mouse Model
16 Female athymic nude CrTac:NCr-Foxn1nu mice were purchased from Taconic Laboratories
(Hudson, NY). 8 mice received subcutaneous injections with 106 U251 MG cancer cells in
Matrigel® (BD Biosciences, San Jose, CA) into each left shoulder and were allowed to grow for
approximately two weeks until the tumors reached 5-10 mm in diameter. Mice were anesthetized
(isoflurane 1.5%, 2 L/min medical air) during tumor implantation and microPET imaging. All
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animal experiments were conducted in accordance with protocols approved by the Institutional
Animal Care and Use Committee of MSK and followed NIH guidelines for animal welfare.
Blood Stability
[18F]-ATRi (approximately 300 μCi, 0.7 Ci/μmol) was injected in healthy athymic nude mice
(n=5) via tail injection. Mice were sacrificed at different time points (0, 60, 120, 180, 240 min
p.i.) and blood was collected. 750 μL of MeCN were added to the collected blood and
centrifuged (5 min at 5000 rpm) to pellet blood cells and proteins. The supernatant was collected
and prepared for HPLC injection by adding 750 μL mQ H2O and filtering. The blood stability
was measured by analytical HPLC analysis (Method B).
Blood Half-Life
The blood half-life of [18F]-ATRi was calculated by measuring the activity of blood samples
collected at different time points (5, 15, 30, 45, 60, 90 and 120 min p.i.). Female nude mice (n =
3) were injected via tail vein with [18F]-ATRi (20% PEG300 / 80% PBS) and blood samples
obtained by retro-orbital bleed using tared capillary tubes. Samples were weighed, and activity
was measured by γ counter. The blood half-life was calculated with Graph Prism 7 (GraphPad
Software, La Jolla, CA) using a two-phase decay least squares fitting method and expressed as
%IA/g.
Chemical Hydrophobicity Index
The Chemical Hydrophobicity Indices (CHI) were measured using a previously developed
procedure [29, 30]. Briefly, reverse phase HPLC was used to measure the retention times of a set
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of standards with known CHI. A standard curve was then created to calculate the CHI of F-
ATRi based on the HPLC retention time.
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Octanol/Water partition coefficient
The lipophilicity of the [18F]-ATRi was acquired by adding 2.5 μCi to a mixture of 0.5 mL of 1-
octanol and 0.5 mL of 25 mM phosphate buffered saline (pH 7.4) and mixed for 5 min. Then, the
mixture was centrifuged at 15.000 rpm for 5 min. 100 μL samples were obtained from organic
and aqueous layers, and the radioactivity of the samples were measured in a γ-counter WIZARD2
automatic γ-counter (PerkinElmer, Boston, MA). The experiment was performed in triplicate,
and the resulting logPo/w was calculated as the mean ± SD.
Plasma protein binding
The plasma protein fraction was determined using the Rapid Equilibrium Dialysis Device
System (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol.
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Membrane dialysis was performed with 500 μM F-ATRi in serum (500 μL) on one side of the
membrane and PBS (700 μL) on the other side. The system was sealed with parafilm and
incubated for 4 h at 37 °C on an orbital shaker set to 100 rpm. Thereafter, 50 μL of solution was
taken from both sides, and samples were treated with 300 μL of precipitation buffer (90/10
acetonitrile/water with 0.1% formic acid) and vortexed to remove protein before HPLC analysis.
After injection, the [18F]-ATRi peaks from each sample were integrated, and the protein bound
fraction determined as %bound= [1 – (Concentration buffer chamber/Concentration plasma
chamber)]x100. The final albumin binding was calculated as the mean ± SD.
Competitive displacement assay
For inhibition studies, U251 MG cells were seeded in a 96 wells plate (105 cells 24 h prior the
experiment). The next day, [18F]-ATRi at a final 0.667 nM concentration was added and co-
incubated together with different Ve-821 concentrations (ranging from 0 to 1333 nM) at 37℃for
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1 h. After 1 h, cells were first washed twice with PBS, then lysed with 1N NaOH, and finally
18
collected and counted in a γ-counter for bound F ligand. The percentage of bound radioligand
at each concentration was measured in triplicate and plotted against Ve-821 concentration.
Competitive displacement curves were fitted using Graph Prism 7 (GraphPad Software Inc, La
Jolla, CA).
In vitro cell uptake
One day prior to the assay, U251 MG cells were placed in 6-well plates (0.5 million cells/well).
On the day of the experiment, cells were washed twice with PBS and further incubated with
[18F]-ATRi (6 μCi/well) at 37℃for 0, 15, 30, 45, 60, 120 and 240 min in MEM medium to allow
for internalization. 100-fold excess of Ve-821 was co-incubated with [18F]-ATRi as blocking
agent for ATR in the blocking study. At each of the reported time points, cells were first washed
twice with PBS and then lysed by incubation with 1M NaOH (10 min at 37℃). Then, the
resulting lysate in each well was collected and the radioactivity measured in a γ-counter.
MicroPET imaging and ex vivo biodistribution
MicroPET imaging studies were performed in subcutaneous U251 MG xenograft bearing
athymic nude mice (n = 8). Mice were divided in two groups (blocked and unblocked) and
administered with [18F]-ATRi (approximately 200 μCi, 0.7 Ci/μmol) via tail vein injection. The
blocked group was pre-injected 30 min before with a 100-fold excess of Ve-821. Approximately
5 min prior to PET acquisition, mice were anesthetized by inhalation of a mixture of isoflurane
(Baxter Healthcare, Deerfield, IL, USA; 2% isoflurane, 2 L/min medical air) and positioned on
the scanner bed. Anesthesia was maintained using a 1% isoflurane/O2 mixture. PET data for each
mouse was recorded starting at 30 min p.i. using dynamic scans of 5 min and acquired for the
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following 90 min. After microPET imaging acquisition was concluded, mice were sacrificed by
CO2 asphyxiation (120 min p.i.) and major organs were collected, weighed, and counted in a γ-
counter. The radiopharmaceutical uptake was expressed as a %ID/g using the following formula:
[(activity in the target organ/grams of tissue)/injected dose]x100% and plotted as the mean ± SD.
Results and Discussion
In this study, we report on the first in vivo [18F]-ATRi ([18F]-9) as a PET imaging agent with
strong similarities in pharmacology, potency, and isoform selectivity to Ve-821, a clinically
18
relevant ATR inhibitor [27]. Structurally, introduction of an F label at the 4-position of the
aromatic ring of Ve-821 (4) appeared to be a viable approach for generating a labeled ATR
inhibitor. Probing whether this functionalization could negatively impact the compound’s
biological activity, we synthesized and profiled the cold fluorinated analogue of our desired
19
probe, F-ATRi 5. The synthesis was readily accomplished and similar to already established
synthetic procedures [31], including some minor modifications (Suppl. Fig. S1 and S2).
Synthesis started from two commercially available precursors, 3-amino-6-bromo-N-phenyl-
pyrazine-2-carboxamide (1a) and 2-(4-methanesulfonylphenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (1b), which were coupled using standard Suzuki reaction conditions [32, 33]. The
reaction yielded an intermediate 2 in 80% yield under microwave-heating at 110 °C for 1 h.
Subsequent reaction with aniline or fluoroaniline in basic conditions yielded the conversion of
the carboxylic acid 3 to the target compound 4 and 5 respectively under microwave-heating at
150 °C for 10 min. Our goal was to develop a reliable synthetic strategy for the routine
production of [18F]-ATRi with sufficiently high isolated radiochemical yields to permit in vivo
studies. As reported by Hendricks and coworkers and Liang et al., direct fluorination of 1,4-
dinitrobenzene with 18F (no carrier added, [n.c.a.]) under phase transfer conditions and
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microwave heating afforded 1-[18F]fluoro-4-nitrobenzene 6 after 5 min at 120 °C [28, 34]. This
intermediate was consecutively reduced by catalytic hydrogenation with sodium borohydride and
Pd/C to yield 4-[18F]fluoroaniline 7 (Fig. 2A and Fig. 2B). The 18F-labeled aniline was
subsequently converted to the desired product [18F]-ATRi ([18F]-9) via coupling with precursor 8
in toluene and after stirring the reaction mixture at RT for 5 min (Fig. 2A) (Suppl. Fig. 3).
Overall, this three-step synthesis is remarkably clean (Fig. 2B) (Suppl. Fig. S4), can be carried
out in less than 2 h, and affords [18F]-9 with an overall isolated decay corrected radiochemical
yield of 30 ± 10 % (after HPLC purification), a radiochemical purity greater than 97% (Fig. 2C)
and a specific activity of 0.7 Ci/μmol (Suppl. Fig. 5). Following scale-up synthesis and
characterization of the radiotracer, we next tested the radiolabeled tracer [18F]-9 both in vitro and
in vivo. Initially, we determined the ex vivo blood stability of [18F]-9 (Fig. 3A and 3B), the in
vivo blood half-life (Fig. 3C), and defined the main pharmacokinetic values for 5 (Fig. 3D). The
ex vivo blood stability of [18F]-9 showed that more than 97% of the tracer was stable 4 h post
injection with no major metabolites observed (Fig. 3B). The blood half-life, following a single
bolus intravenous injection via tail vein, showed a biphasic pharmacokinetic profile with rapid
elimination of [18F]-9 during the first 10 min, similarly to other small molecule inhibitors [28,
35]. The weighted blood half-life was determined to be 6.2 min. Contrary to the rapid initial
elimination, redistribution of the agent is slow, which is likely due to ATR being expressed in
immune cells, which would consequently lead to specific retention of the tracer, explaining the
observed plateau [9, 11, 36]. The basic pharmacokinetic profile of 5 demonstrated that, similarly
to Ve-821, 19F-ATRi has a lipophilic behavior, as proven by its CHI value (76.8) and high
plasma protein binding (Plasma Free Fraction ~ 10%). The LogPO/W of the radiofluorinated
19
[18F]-9 tracer was measured to be 1.6, corroborating the CHI values obtained with F-ATRi
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(Fig. 3D). Together with multiple studies reporting expression of ATR in glioma cell lines [37-
40] we chose U251 MG, a cell line with high ATR mRNA expression [41, 42], as a mouse
model for our in vitro/in vivo studies. The mRNA level of ATR in GBM, although significantly
higher in comparison to healthy brain tissues [41, 42], are lower than other major solid tumors
such as squamous cell lung, breast, colorectal, pancreatic, prostate, and skin cancer, as well as
most of the remaining cancer types (Suppl. Fig. 6). This is corroborated by Nadkarni et al. [39]
and Ramirez et al. [40] who measured ATR downstream signaling and ATR expression in U251
MG cells. Both groups used glioblastoma cell lines as a model to study the effect of ATM/ATR
inhibitors. Further, we showed the possibility to use Ve-821 to study the in vitro cell uptake of
[18F]-9. The ability to suppress [18F]-9 uptake by competitive displacement in U251 MG cells
was an indication of the similar inhibition effect between Ve-821 and [18F]-9 (Fig. 4A). In fact,
the tracer appears to have an affinity for ATR comparable to Ve-821 (competitive displacement
value of [18F]-ATRi by Ve-821 = 38 nM). Cell uptake rates were also significantly diminished in
U251 MG cells blocked by co-incubation with Ve-821 (3.5 ± 0.1 % at 240 min incubation with
[18F]-ATRi only vs. 0.16 ± 0.1 % in U251 MG cells blocked with Ve-821 and co-incubated with
[18F]-ATRi for 240 min) (Fig. 4B). These results taken together suggest that replacement of the
para proton on the aniline ring with an 18F atom did not fundamentally perturb the binding
properties of [18F]-9 in comparison to Ve-821 (Ve-821: IC50 = 26 nM, [24]) and that Ve-821 and
[18F]-9 presumably bind the same target. However, a more accurate and thorough IC50 study
should be performed to determine the real inhibition of the ATR target and thus the measure of
the activity of [18F]-9 in the presence of a known concentration of ATP. The uptake (%ID/g) and
the biodistribution of [18F]-9 are shown in Fig. 5A-C and Suppl. Fig. S7. For each experiment,
two groups of mice were sacrificed at 2 h following intravenous injection of the probe. Tissues
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were then excised, weighed and 18F activity was measured. [18F]-9 had the highest concentration
in the kidney, liver, intestines and blood, with relatively low distribution in other tissues (Fig. 5B
and Fig 5C). The significant blocking effect in blood (from 1.85 ± 0.47 to 0.84 ± 0.30 %ID/g)
supports our hypothesis that [18F]-9 is retained in the blood pool selectively, rather than absorbed
non-specifically by blood plasma components. [18F]-9 was also observed to specifically
accumulate in tumors as proven by TAC (Fig. 5B) and blocking experiment (from 1.48 ± 0.39 to
0.46 ± 0.12 %ID/g) (Fig. 5C and Suppl. Fig. S7). The tracer washes out via the hepatobiliary
route as evident by microPET image reconstruction and biodistribution data. Kidney and liver
showed uptake values of 3.51 ± 0.23 and 3.68 ± 1.39 %ID/g, respectively, in biodistribution
experiments. As expected for selective competition, a high tumor to background ratio was
observed in most of the non-targeting tissues (Fig. 5D). This tracer shows remarkable tumor-to-
brain ratios (>15), leaning evidence towards the possibility that [18F]-9 could be used for brain
tumor imaging. In conclusion, our data support that [18F]-ATRi could be a great resource to
image ATR, together with all its implications for better understanding ATR therapy, target
engagement and treatment monitoring.
Conclusions
Here we report a novel fluorinated [18F]-ATR inhibitor ([18F]-9) that was synthesized starting
from Ve-821, a clinically relevant ATR inhibitor. [18F]-9 was obtained in good yield
18
(radiochemical corrected yield ∼ 30% overall), and was efficiently labeled with F-fluoride (<
2 h; > 97% radiochemical purity). Our data suggest that [18F]-ATRi is the first in vivo ATR PET
imaging agent potentially leading to better understanding of ATR therapy, target engagement
and treatment monitoring. This pilot study thus shows proof of principle that an imaging agent
such as [18F]-ATRi could be a first step towards a clinically translatable diagnostic tool. It
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encourages further investigations with the ultimate aim of non-invasively sensing and
quantifying ATR kinase.
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Figures
Fig. 1. ATM and ATR are activated by DNA damage as well as DNA replication stress. ATR
triggers Chk1 and other downstream targets to promote DNA repair, stabilization, restart of
stalled replication forks and transient cell cycle arrest.
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B
4-[18F]fluoronitrobenzene (6)
4-[18F]fluoroaniline (7)
A
[
18F]-ATRi ([18F]-9)
100
50
0
NO2
O2N
O2N
[
18F]F-, DMSO, MW, 120℃, 3 min
18F
6
0
5
10
minutes
15
20
Pd/C, NaBH4, MeOH, rt, 7 min
18F
C
NH2
N
O
18F
100
50
0
NH2
O
[
18F]-ATRi ([18F]-9)
7
N
Cl
H2N
N
N
H
N
toluene, 5 min, rt
0
10
minutes
20
1x106
5x104
0
[
19F]-ATRi (5)
O
S
O
O
S
O
9
8
0
20
10
minutes
Fig. 2. Synthesis and analysis of [18F]-ATRi. (A) Shows the radiochemical synthesis of [18F]-
ATRi. 4-[18F]fluoroaniline (7) was obtained by the reduction of 4-[18F]fluoronitrobenzene (6) in
the presence of Pd/C and NaBH4.. Following, 4-[18F]fluoroaniline was coupled with the acid
chloride precursor (8) to obtain [18F]-ATRi (9) (room temperature for 5 min); (B) analytical
radioactivity traces of the three reaction step with relative products: 4-[18F]fluoronitrobenzene
(6), 4-[18F]fluoroaniline (7) and [18F]-ATRi (9). (C) Quality control shows that the radiochemical
purity for [18F]-ATRi (9) is > 97% and perfectly coelutes with the cold reference synthesized
compound 19F-ATRi (5).
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0 min
60 min
A
B
C
120 min
15
180 min
240 min
100
50
0
10
5
0
120
0
20
0
60
minutes
minutes
D
Time Point
0 minutes
Stability (%)
>97%
CHI
76.77
1.6
60 minutes
120 minutes
180 minutes
240 minutes
>97%
>97%
LogP(o/w)
>97%
>97%
Plasma Free Fraction (%)
9.2 ± 2.8
Fig. 3. Stability and basic pharmacokinetics of [18F]-ATRi and 19F-ATRi. (A) Radiochemical
stability of [18F]-ATRi. Blood was collected from healthy mice after injection of [18F]-ATRi (200
μCi/mouse). 5 time points after tracer i.v. injection were evaluated (0, 60, 120, 180, 240
minutes). (B) Tabulation of tracer stability after 4 hours. (C) In vivo blood half life of [18F]-
ATRi. Healthy mice (n=4) were injected with [18F]-ATRi and blood collected at different time
points. Results are plotted as % injected activity/g over 120 minutes as a 2-phase decay curve
indicating a fast clearance from bloodstream (< 10 minutes). (D) Lipophilicity profiling and
Plasma Free Fraction of 19F-ATRi and [18F]-ATRi.
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A
B
4
[18F]-ATRi
100
U251
3
2
50
0
1
0
Ve-821/[18F]-ATRi
-2
-1
0
1
2
3
0
60
120
180
240
log conc (nM)
minutes
Fig. 4. Competitive displacement of [18F]-ATRi by Ve-821 and [18F]-ATRi cell uptake. A)
Shows the displacement for [18F]-ATRi by increasingly high concentrations of Ve-821 (IC50 = 26
nM) in U251 MG cells (n=3). (B) Shows the uptake rate of [18F]-ATRi in a blocked vs. non-
blocked cell uptake study. Cells were incubated for a maximum of 240 minutes with [18F]-ATRi
only or with [18F]-ATRi + Ve-821, washed twice and lysed. The collected activity was quantified
in a γ-counter. Results indicate a specific blocking effect of cells co-incubated with the ATR
inhibitor Ve-821 (n=4).
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A
B
20
[18F]-ATRi
Ve-821/[18F]-ATRi
6.0
Tumor
Liver
Kidney
Muscle
Heart
15
Brain
10
5
0
0
0
40
60
20
80
minutes
C
D
25
20
15
10
5
15
10
5
[18F]-ATRi
Ve-821/[18F]-ATRi
0
0
.
t
r
a
r
.
t
t
.
.
e
r
r
g
s
h
a
t
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e
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s
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l
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u
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p
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l
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m
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P
S
L
S
S
L
Fig. 5. MicroPET imaging and ex vivo biodistribution. (A) Representative coronal microPET
image of U251 MG bearing-mouse after injection of [18F]-ATRi (unblocked, left panel) and after
pre-injection of blocking agent Ve-821 / [18F]-ATRi (100-fold excess, 30 min before, right
panel). (B) Shows %ID/g calculated from ROI activity curves (5 minutes time frames over a 90
min dynamic scan) for tumor, liver, kidney, muscle, heart and brain. (C) Ex vivo biodistribution
of [18F]-ATRi. [18F]-ATRi was tested in U251 MG mice xenografts sacrificed at 120 min p.i..
Effects of blocking were observed after injection of 100-fold excess of Ve-821 (n = 4 per group).
Values are plotted as %ID/g (n=3). (D) Tumor to non-tumor ratios.
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AUTHOR INFORMATION
Corresponding Author
* Corresponding author: T. Reiner, PhD.
Email: reinert@mskcc.org
Present Addresses
† Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New
York, NY 10065.
Author Contributions
G.C and T.R. designed the experiments and analyzed the data. G.C., B.C., J.T., A.S., and A.V.
carried out the experiments. G.C. and T.R. interpreted data and wrote the manuscript. All authors
read, provided feedback on, and approved the manuscript.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
The authors thank Dr. Jason S. Lewis, Dr. NagaVara Kishore Pillarsetty and Dr. Edmund J.
Keliher for helpful discussions, Dr. Christian Brand for help with experiments and Dave Gregory
for editing the manuscript. Technical services provided by the MSK Small-Animal Imaging Core
Facility, supported in part by NIH Cancer Center Support Grant No 2 P30 CA008748-48, are
gratefully acknowledged. The authors thank the NIH (K25 EB016673, R01 CA204441 and R21
CA191679 for T.R.), the Brain Tumor Center of Memorial Sloan-Kettering Cancer (for T.R.),
the Center for Molecular Imaging and Nanotechnology (for T.R.) and the American-Italian
Cancer Foundation (AICF) Post-Doctoral Research Fellowship (for G.C.) for their generous
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support. The Tow Foundation Fellowship Program in Molecular Imaging and Nanotechnology
(for B.C.), the National Science Foundation's Integrative Graduate Education and Research
Traineeship (IGERT 0965983 at Hunter College for B.C.).
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REFERENCES
[1] Jackson SP and Helleday T. Drugging DNA repair. Science 2016;352:1178-9.
[2] Da-Rè C and Halazonetis TD. DNA replication stress as an Achilles’ heel of cancer.
Oncotarget; Vol 6, No 1 2015.
[3] Bartek J, Mistrik M, and Bartkova J. Thresholds of replication stress signaling in cancer
development and treatment. Nat Struct Mol Biol 2012;19:5-7.
[4] Jackson SP and Bartek J. The DNA-damage response in human biology and disease.
Nature 2009;461:1071-8.
[5] Kastan MB and Bartek J. Cell-cycle checkpoints and cancer. Nature 2004;432:316-23.
[6] Benada J and Macurek L. Targeting the Checkpoint to Kill Cancer Cells. Biomolecules
2015;5.
[7] Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev
2001;15:2177-96.
[8] Manic G, Obrist F, Sistigu A, and Vitale I. Trial Watch: Targeting ATM–CHK2 and ATR–
CHK1 pathways for anticancer therapy. Molecular & Cellular Oncology 2015;2:e1012976.
[9] Weber AM and Ryan AJ. ATM and ATR as therapeutic targets in cancer. Pharmacology &
therapeutics 2014.
[10] Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD, et
al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature
2010;463:191-6.
[11] Karnitz LM and Zou L. Molecular Pathways: Targeting ATR in Cancer Therapy. Clinical
Cancer Research 2015.
[12] Rodríguez-Bravo V, Guaita-Esteruelas S, Salvador N, Bachs O, and Agell N. Different S/M
Checkpoint Responses of Tumor and Non–Tumor Cell Lines to DNA Replication Inhibition.
Cancer Research 2007;67:11648-56.
[13] Ruzankina Y, Schoppy DW, Asare A, Clark CE, Vonderheide RH, and Brown EJ. Tissue
regenerative delays and synthetic lethality in adult mice after combined deletion of Atr and
Trp53. Nat Genet 2009;41:1144-9.
[14] Ruzankina Y, Pinzon-Guzman C, Asare A, Ong T, Pontano L, Cotsarelis G, et al. Deletion
of the Developmentally Essential Gene ATR in Adult Mice Leads to Age-Related Phenotypes
and Stem Cell Loss. Cell Stem Cell 2007;1:113-26.
[15] Cimprich KA and Cortez D. ATR: an essential regulator of genome integrity. Nat Rev Mol
Cell Biol 2008;9:616-27.
[16] Casper AM, Nghiem P, Arlt MF, and Glover TW. ATR Regulates Fragile Site Stability. Cell
2002;111:779-89.
[17] Kwok M, Davies N, Agathanggelou A, Smith E, Oldreive C, Petermann E, et al. ATR
inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective
chronic lymphocytic leukemia cells. Blood 2016;127:582-95.
[18] Abdel-Fatah TMA, Middleton FK, Arora A, Agarwal D, Chen T, Moseley PM, et al.
Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target
validation in breast cancer. Molecular Oncology 2015;9:569-85.
[19] Huntoon CJ, Flatten KS, Wahner Hendrickson AE, Huehls AM, Sutor SL, Kaufmann SH, et
al. ATR Inhibition Broadly Sensitizes Ovarian Cancer Cells to Chemotherapy Independent of
BRCA Status. Cancer Research 2013;73:3683-91.
[20] Brown EJ and Baltimore D. Essential and dispensable roles of ATR in cell cycle arrest and
genome maintenance. Genes Dev 2003;17:615-28.
[21] Ahmed SU, Carruthers R, Gilmour L, Yildirim S, Watts C, and Chalmers AJ. Selective
Inhibition of Parallel DNA Damage Response Pathways Optimizes Radiosensitization of
Glioblastoma Stem-like Cells. Cancer Research 2015;75:4416-28.
24

ACCEPTED MANUSCRIPT
[22] Josse R, Martin SE, Guha R, Ormanoglu P, Pfister TD, Reaper PM, et al. The ATR
inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase I inhibitors by disabling
DNA replication initiation and fork elongation responses. Cancer Research 2014.
[23] Prevo R, Fokas E, Reaper PM, Charlton PA, Pollard JR, McKenna WG, et al. The novel
ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and
chemotherapy. Cancer Biology & Therapy 2012;13:1072-81.
[24] Reaper PM, Griffiths MR, Long JM, Charrier J-D, MacCormick S, Charlton PA, et al.
Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol
2011;7:428-30.
[25] Li C-C, Yang J-C, Lu M-C, Lee C-L, Peng C-Y, Hsu W-Y, et al. ATR-Chk1 signaling
inhibition as a therapeutic strategy to enhance cisplatin chemosensitivity in urothelial bladder
cancer. Oncotarget; Vol 7, No 2 2015.
[26] Lerner RG, Grossauer S, Kadkhodaei B, Meyers I, Sidorov M, Koeck K, et al. Targeting a
Plk1-Controlled Polarity Checkpoint in Therapy-Resistant Glioblastoma-Propagating Cells.
Cancer Research 2015;75:5355-66.
[27] Charrier J-D, Durrant SJ, Golec JMC, Kay DP, Knegtel RMA, MacCormick S, et al.
Discovery of Potent and Selective Inhibitors of Ataxia Telangiectasia Mutated and Rad3 Related
(ATR) Protein Kinase as Potential Anticancer Agents. Journal of Medicinal Chemistry
2011;54:2320-30.
[28] Hendricks JA, Keliher EJ, Marinelli B, Reiner T, Weissleder R, and Mazitschek R. In Vivo
PET Imaging of Histone Deacetylases by 18F-Suberoylanilide Hydroxamic Acid (18F-SAHA).
Journal of Medicinal Chemistry 2011;54:5576-82.
[29] Valkó K. Application of high-performance liquid chromatography based measurements of
lipophilicity to model biological distribution. Journal of Chromatography A 2004;1037:299-310.
[30] Valkó K, Bevan C, and Reynolds D. Chromatographic Hydrophobicity Index by Fast-
Gradient RP-HPLC:ꢀ A High-Throughput Alternative to log P/log D. Analytical Chemistry
1997;69:2022-9.
[31] Valeur E and Bradley M. Amide bond formation: beyond the myth of coupling reagents.
Chemical Society Reviews 2009;38:606-31.
[32] Sindhuja E, Ramesh R, and Liu Y. Palladium(ii) thiocarboxamide complexes: synthesis,
characterisation and application to catalytic Suzuki coupling reactions. Dalton Transactions
2012;41:5351-61.
[33] Thakur A, Zhang K, and Louie J. Suzuki-Miyaura coupling of heteroaryl boronic acids and
vinyl chlorides. Chemical Communications 2012;48:203-5.
[34] Liang SH, Collier TL, Rotstein BH, Lewis R, Steck M, and Vasdev N. Rapid microfluidic flow
hydrogenation for reduction or deprotection of 18F-labeled compounds. Chemical
Communications 2013;49:8755-7.
[35] Carney B, Carlucci G, Salinas B, Di Gialleonardo V, Kossatz S, Vansteene A, et al. Non-
invasive PET Imaging of PARP1 Expression in Glioblastoma Models. Mol Imaging Biol
2016;18:386-92.
[36] Bauer M, Goldstein M, Heylmann D, and Kaina B. Human Monocytes Undergo Excessive
Apoptosis following Temozolomide Activating the ATM/ATR Pathway While Dendritic Cells and
Macrophages Are Resistant. PLoS One 2012;7:e39956.
[37] Kao GD, Jiang Z, Fernandes AM, Gupta AK, and Maity A. Inhibition of Phosphatidylinositol-
3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells following Ionizing
Radiation. Journal of Biological Chemistry 2007;282:21206-12.
[38] Rivera M, Sukhdeo K, and Yu JS. Ionizing Radiation in Glioblastoma Initiating Cells.
Frontiers in Oncology 2013;3.
[39] Nadkarni A, Shrivastav M, Mladek AC, Schwingler PM, Grogan PT, Chen J, et al. ATM
inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM
cells. Journal of Neuro-Oncology 2012;110:349-57.
25

ACCEPTED MANUSCRIPT
[40] Ramirez YP, Mladek AC, Phillips RM, Gynther M, Rautio J, Ross AH, et al. Evaluation of
Novel Imidazotetrazine Analogues Designed to Overcome Temozolomide Resistance and
Glioblastoma Regrowth. Molecular Cancer Therapeutics 2015;14:111.
[41] Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, et al. Tissue-
based map of the human proteome. Science 2015;347.
[42] Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, et al. Towards a
knowledge-based Human Protein Atlas. Nat Biotech 2010;28:1248-50.
26