Synthesis and evaluation of an AZD2461 [18F]PET probe in non-human primates reveals the PARP-1 inhibitor to be non-blood-brain barrier penetrant

Article abstract of DOI:10.1016/j.bioorg.2018.10.015

Poly(ADP-ribose)polymerase-1 inhibitor (PARPi) AZD2461 was designed to be a weak P-glycoprotein (P-gp) analogue of FDA approved olaparib. With this chemical property in mind, we utilized the AZD2461 ligand architecture to develop a CNS penetrant and PARP-1 selective imaging probe, in order to investigate PARP-1 mediated neuroinflammation and neurodegenerative diseases, such as Alzheimer's and Parkinson's. Our work led to the identification of several high-affinity PARPi, including AZD2461 congener 9e (PARP-1 IC₅₀ = 3.9 ± 1.2 nM), which was further evaluated as a potential ¹⁸F-PET brain imaging probe. However, despite the similar molecular scaffolds of 9e and AZD2461, our studies revealed non-appreciable brain-uptake of [¹⁸F]9e in non-human primates, suggesting AZD2461 to be non-CNS penetrant.

Full text of DOI:10.1016/j.bioorg.2018.10.015

BioorganicChemistry83(2019)242–249
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of an AZD2461 [18F]PET probe in non-human
primates reveals the PARP-1 inhibitor to be non-blood-brain barrier
penetrant
Sean W. Reillya, Laura N. Puentesb, Alexander Schmitza, Chia-Ju Hsieha, Chi-Chang Wenga,
Catherine Houa, Shihong Lia, Yin-Ming Kuoa, Prashanth Padakantia, Hsiaoju Leea,
⁎
⁎
Aladdin A. Riada, Mehran Makvandia, , Robert H. Macha,
a
Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
b
Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Poly(ADP-ribose)polymerase-1 inhibitor (PARPi) AZD2461 was designed to be a weak P-glycoprotein (P-gp)
analogue of FDA approved olaparib. With this chemical property in mind, we utilized the AZD2461 ligand
architecture to develop a CNS penetrant and PARP-1 selective imaging probe, in order to investigate PARP-1
mediated neuroinflammation and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Our work
led to the identification of several high-affinity PARPi, including AZD2461 congener 9e (PARP-1
PARP-1 inhibitor
MicroPET imaging
P-glycoprotein
Blood-brain barrier
AZD2461
IC50 = 3.9
1.2 nM), which was further evaluated as a potential 18F-PET brain imaging probe. However, de-
spite the similar molecular scaffolds of 9e and AZD2461, our studies revealed non-appreciable brain-uptake of
[
18F]9e in non-human primates, suggesting AZD2461 to be non-CNS penetrant.
1. Introduction
the drug-efflux pump in mouse models [11], resulting in AstraZeneca to
develop AZD2461 (2), a poor P-gp substrate, as a potential backup.
Since then, 2 [12], veliparib (3) [10] and BGB-290 (4) [13] are among
the few PARP-1 inhibitors (PARPi) to be reported as weak P-gp sub-
strates (Fig. 1). With this chemical property in mind, we evaluated a
structurally similar congener of AZD2461 as a potential blood-brain
barrier (BBB) penetrable PET probe. Herein, we report the synthesis and
PARP-1 binding profiles of AZD2461 analogues, as well as PARP-1
specificity and brain uptake of compound [18F]9e using in vitro auto-
radiography and microPET imaging studies, respectively.
Poly(ADP-ribose) polymerase-1 is a nuclear protein that plays an
integral role in numerous physiological cellular mechanisms including
DNA damage repair, maintenance of genomic stability, and apoptosis
[1]. As such, this protein has become an attractive therapeutic target for
cancer [2,3], neuroinflamation and neurodegeneration [4], cardiovas-
cular disease [5], and even drug addiction [6,7]. Moreover, PARP-1
mediated cell death, termed parthanatos, is the second most studied
form of cell death and plays a key role in several neurodegenerative
diseases [8]. Consequently, a PARP-1 positron emission tomography
(PET) neuroimaging probe would advance our limited understanding in
the neurodegenerative pathways associated with PARP-1, and bring
new insight into developing therapeutic strategies to combat these
deadly neurological diseases. Unfortunately, all known PARP-1 imaging
agents have been shown to be non-CNS penetrant [9].
2. Results and discussion
2.1. Chemistry
Initial synthesis of AZD2461 fluoroethoxy analogues began with
treating commercially available hydroxyl amines (5a-f) with NaH, fol-
lowed by alkylating reagent 6 [14] in DMF (Scheme 1). Boc-deprotec-
tion of the O-alkylated synthons was achieved with CF3COOH,
Despite the recent advances in PARP-1 inhibitor (PARPi) develop-
ment [2], there have been limited studies illustrating P-gp status of
PARPi [10]. In 2008, olapraib (1) was shown to induce expression of
Abbreviations: ATP, adenosine triphosphate; BBB, blood-brain barrier; EDC HCl, ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; MDR, multidrug
resistance, P-gp, permeability glycoprotein; TEA, triethylamine; THF, tetrahydrofuran
⁎
Corresponding authors.
E-mail addresses: makvandi@pennmedicine.upenn.edu (M. Makvandi), rmach@mail.med.upenn.edu (R.H. Mach).
https://doi.org/10.1016/j.bioorg.2018.10.015
Received 12 July 2018; Received in revised form 8 October 2018; Accepted 9 October 2018
Availableonline17October2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

S.W. Reilly et al.
BioorganicChemistry83(2019)242–249
affording intermediates 7a-f. Target compounds 9a-f were acquired by
crude coupling of the desired fluoroethoxy free-amine (7a-f) with 8,
using reagents HOBt and EDC in THF. These coupling conditions were
also utilized to prepare 11a-b from synthons 8 and 10a-b.
Following our previously reported 20 min C–N cross-coupling pro-
tocol [15], diazaspiro moiety 13 was obtained under aerobic conditions
for development of 14. Synthesis of azaspiro motif 16 began with
treating 5a with triphenylphosphine and carbon tetrabromide in THF to
access intermediate 15. Coupling bromide synthon 15 and (4-fluor-
ophenyl)boronic acid, using a modified Ni-catalyzed Suzuki reaction
reported by Fu and co-workers [16], allowed access to 16. Finally, 16
was reacted with penultimate compound 8 to afford 17 good yield.
2.2. Biological evaluation
2.2.1. PARP-1 radioligand binding
Binding profiles of compounds 2, 9a-f, 11a-b, 14, and 17 were
obtained following our previously reported radioligand binding assay
conducted in BRCA1 methylated ovarian cancer cells (OVCAR8) [17].
PARP-1 enzymatic inhibition and cLogP values of AZD2461 analogues
containing O-alkylated fluoroethoxy nitrogen heterocycles were eval-
uated (9a-f) and outlined in Table 1. In terms of PARP-1 IC50, we
identified compound 9e to be the most promising candidate in the
Fig. 1. Chemical structures of reported CNS-penetrant PARP-1 inhibitors.
fluoroethoxy series, with
a comparable enzymatic inhibition to
AZD2461 parent compound 2. Next, we examined the binding prop-
erties of more rigid PARPi analogues (11a-b, 14, 17), each containing a
fluorinated-aryl ring system. Compared to the IC50 profiles of 9a-f and
AZD2461, we found these ligands to be more potent inhibitors of PARP-
1 enzymatic activity. Compound 14, containing a 2,6-diazaspiro[3.3]
heptane core, was found to be the most potent PARPi in this study, a
property also observed in our recent report describing the unique
pharmacological properties afforded when implementing this piper-
azine bioisostere in the olaparib architecture [18]. However, for the
purposes of this study, we furthered our investigation with 9e, due to
the structural similarity of the compound and AZD2461, as well as the
PARP-1 IC50 binding data (3.9 nM) and the favorable estimated cLogP
value (∼2.2) [19].
Table 1
PARP-1 IC50 Values for 9a-f, 11a-b, 14, and 17.a
cLogPc
2.07
b
Compound
Coupled amine
PARP-1 IC50
2
2.8
1.1
1.1
1.1
9a
10.7
2.49
9b
7.5
2.04
9c (R)
9d (S)
9e
10.1
6.8
1.1
2.15
2.15
2.26
1.1
1.2
3.9
9f
5.6
1.1
2.71
11a (Y = N)
0.6
1.3
1.2
NA
3.37
3.64
11b (Y = CH)
14 (Y = N)
17 (Y = CH)
0.3
2.8
1.2
NA
3.24
3.40
Scheme 1. Synthesis of PARPi 9a-f, 11a-b, 14, and 17a. aReagents and con-
ditions: (i) Respective hydroxyl amine (5a-f), NaH, 6, DMF, rt, 2 h; (ii) TFA,
DCM, rt, 3 h; (iii) 8, respective free-amine, HOBt hydrate, EDC HCl, TEA, THF,
60 °C, 12 h. (iv) 5a, PPh3, CBr4, THF, rt; (v) 15, (4-fluorophenyl)boronic acid,
NiI2, trans-2-aminocyclohexanol hydrochloride, NaHMDS, 2-methyl-2-butanol
80 °C, 12 h.
a
IC50 (nM)
(SEM) values represent inhibition of PARP-1 enzymatic ac-
tivity using our previously reported radioligand binding methodology (Ref.
[17]).
b
Dose response curves were produced to calculate 50% maximum inhibition
values (IC50) where n = 3.
c
Calculated using ChemDraw Professional 15.1.
243

S.W. Reilly et al.
BioorganicChemistry83(2019)242–249
1.5
1.0
0.5
0.0
l
i
m
b
e
b
4
7
1
O 4
i
9
1
1
r
1
a
V
a
p
a 3
p
a
a
l
N
r
o
e
v
Fig. 2. Luminescence generated with 9e, 11b, 14, and 17, olaparib, Verapamil,
and Na3VO4. Verapamil, a substrate for P-gp that stimulates P-gp ATPase ac-
tivity resulting in decreased luminescence, was utilized as a positive control.
Increase in luminescence results from the light-generating reaction from luci-
ferase and unmetabolized ATP. Unconsumed ATP indicates a decrease in P-gp
ATPase stimulation, rendering the compound as a P-gp inhibitor. Data is nor-
malized to known P-gp inhibitor Na3VO4.
Fig. 3. In vitro autoradiography blocking study conducted with [18F]9e and
olaparib.
achieved in a facile one-step radiolabeling strategy using precursor 19a.
Although not optimized, this method afforded a radiochemical yield of
8–12% for [18F]9e, with a specific activity of 8,491 Ci/mmol (n = 2).
The mesylate precursor 19b was also evaluated, however, low radio-
chemical yields were obtained (∼1–3%).
2.2.2. P-gp assay
Compound 9e, and select compounds, were examined in a P-gp
assay to determine if these PARPi act as stimulators or inhibitors of the
plasma membrane protein (Fig. 2). Luminescence generated from un-
metabolized ATP was measured after incubating the compounds with
recombinant human P-gp, an adenosine (ATP)-dependent drug efflux
pump. Compounds acting as stimulators of P-gp ATPase, resulted in
decreased luminescence due to the metabolized ATP. Similar lumines-
cence signals were triggered with both positive control verapamil and
PARPi olaparib, known substrates for P-gp [12]. In contrast, increased
luminescence was observed for known P-gp inhibitor Na3VO4 and 9e,
11b, 14, and 17 further indicating AZD2461 congener 9e to be a sui-
table brain imaging probe candidate.
2.2.4. In vitro autoradiography
In vitro autoradiography was performed with brain sections from
Balb/c mice to verify binding specificity of radiolabeled compound
[
18F]9e (Fig. 3). We found the distribution of [18F]9e to be mostly
conserved in the cortex, hippocampus, and cerebellum. Olaparib was
utilized as the blocking agent to screen any non-specific binding of the
radioligand.
2.2.5. MicroPET imaging
Small animal PET imaging studies using rhesus macaques were
conducted with compound [18F]9e and [18F]FTT (Fig. 4), a PARP-1 PET
tracer under clinical investigation [20] known to be a Pg-p substrate.
Despite possessing similar chemical structure and PARP-1 affinity as
AZD2461, no appreciable brain uptake of [18F]9e was observed in any
of the brain regions (Fig. 5A). These images are similar to the those
obtained and expected with [18F]FTT (Fig. 5B), showing the PARPi to
be non-BBB penetrable. Metabolic stability of [18F]9e was assessed
using the monkey blood from each imaging study and found 42% and
45% of the parent compound intact at 40 min and 37 min, respectively
(See SI).
2.2.3. Radiochemistry
Radiolabeling precursors for [18F]9e were initially prepared by
coupling commercially available compounds 8 with 2-(piperidin-4-
yloxy)ethan-1-ol, in the presence of HOBt, EDC, TEA in THF, to obtain
intermediate 18 in moderate yield (Scheme 2). Next, 18 was reacted
with 4-toluenesulfonyl chloride or methanesulfonyl chloride to afford
precursors 19a and 19b, respectively. Access to [18F]9e was then
Rodent studies were then performed with [18F]9e, including control
and P-gp knockout (k/o) mice models (data not shown), to validate the
tracer as a non-P-gp substrate. However, analysis from select rodent
studies showed [18F]9e to be non-metabolically stable, with over 80%
decomposition of the parent compound in both mice blood and brain in
just 5 min (See SI).
Scheme 2. Radiosynthesis of [18F]9ea. aReagents and conditions: (i) 8, 2-(pi-
peridin-4-yloxy)ethan-1-ol, HOBt hydrate, EDC HCl, TEA, THF, 60 °C, 12 h; (ii)
TsCl or MsCl, TEA, DCM, 12 h; (iii) [18F]KF, K222, K2CO3, DMSO, 120 °C,
20 min.
Fig. 4. Chemical structure of [18F]FTT PET probe.
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S.W. Reilly et al.
BioorganicChemistry83(2019)242–249
Fig. 5. Monkey PET images indicating the low brain uptake of [18F]9e (A) and [18F]FTT (B) indicated by SUV.
3. Conclusion
614 and 1315 were synthesized following previously reported condi-
tions. NMR spectra were taken on a Bruker DMX 500 MHz. Compound
structures and identity were confirmed by 1H and 13C NMR, and mass
spectroscopy. Compound purity greater than 95% was determined by
LCMS analysis using a 2695 Alliance LCMS. All other commercial re-
agents were purchased and used without further purification. Pur-
ification of organic compounds were carried out on a Biotage Isolera
One with a dual-wavelength UV–VIS detector. Chemical shifts (δ) in the
NMR spectra (1H and 13C) were referenced by assigning the residual
solvent peaks.
The goal of this study was to develop derivatives of AZD2461 for
PARP-1 neuroimaging applications. We disclosed the synthesis and
evaluation of AZD2461 analogues, including two novel PARPi con-
taining spiro[3.3]heptane cores (14 and 17) with excellent PARP-1
inhibition (0.3 nM and 2.8 nM, respectively). We identified AZD2461
congener 9e as a potent PARP-1 inhibitor (3.9 nM), and a weak sub-
strate for P-gp. Radioligand [18F]9e was then examined as a potential
PARP-1 PET neuroimaging probe, however, [18F]9e demonstrated no
brain uptake in our monkey PET imaging studies. We further in-
vestigated [18F]9e in rodent models, including control and P-gp
knockout (k/o) mice models, but found over 80% of the parent com-
pound to be metabolized. Thus, it cannot be ruled out [18F]9e may
indeed be a substrate for other clinically relevant MDR pumps [21],
perhaps explaining the non-appreciable brain uptake observed in our
non-human primate studies. Current evaluation is ongoing to identify a
more metabolically stable PARPi that is a suitable candidate for PET
neuroimaging.
4.1.2. General synthetic procedure for compounds 7a-f
NaH 60% dispersion in mineral oil (3.3 mmol) was slowly added to
a stirring solution of hydroxyl amine (5a-f) (3.0 mmol) in 6 mL of DMF
at room temp, and stirred for 30 min. Compound 6 was then slowly
added to reaction mixture, followed by an additional hour of stirring. A
saturated NaHCO3 (aq) solution (40 mL) was then added to the crude
reaction mixture and stirred at room temp for 1 h. The reaction mixture
was extracted with CH2Cl2 (3 × 20 mL) to afford the crude product. The
subsequent residue was loaded onto a Biotage SNAP flash purification
cartridge, eluting with a 2:5 EtOAc/hexanes gradient, to afford the
desired Boc-protected O-alkylated fluoroethoxy compounds, identified
by LCMS. The intermediate was then dissolved in CH2Cl2 (2 mL), fol-
lowed by dropwise addition of CF3COOH (2 mL), and stirred at room
temperature for 3 h. Volatiles were then removed under reduced pres-
sure and the crude product was neutralized with a saturated NaHCO3
(aq) solution (10 mL). The reaction mixture was extracted with CH2Cl2
(3 × 20 mL), and the organic layers were combined, dried, and
4. Experimental
4.1. Chemistry
4.1.1. General experimental information
Chemical compounds 5a-f, 8, and 10a-b were purchased and used
without further purification. Compound 8 can also be prepared fol-
lowing previously reported literature conditions [22,23]. Intermediate
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S.W. Reilly et al.
BioorganicChemistry83(2019)242–249
concentrated to afford the free-amine intermediates (7a-f) as oils used
rotamers) δ 165.1 (2xC), 161.1, 156.3 (2xC) (d, J(1)C–F = 248.0 Hz;
in the next step without any further purification.
J
J
J
J
(2)C–F = 247.6 Hz), 145.7, 134.2 (d, JC–C–C–C–F = 3.7 Hz), 134.1 (d,
C–C–C–C–F = 3.7 Hz), 133.6, 131.5, 131.4 (2xC) (d, J(1)C–C–C–F = 8.6 Hz;
(2)C–C–C–F = 8.1 Hz), 129.6, 129.1 (d, JC–C–C–F = 3.8 Hz), 128.9 (d,
C–C–C–F = 4.0 Hz), 128.3, 127.0, 125.4 (d, JC–C–F = 17.7 Hz), 125.4
4.1.3. General synthetic procedure for compounds 9a-f, 11a-b, and 14
The desired free-amine intermediate (7a-f, 10a-b, or 13)
(1.0 mmol), 8 (1.0 mmol), HOBt hydrate (1.0 mmol), EDC hydro-
chloride (1.0 mmol), and Et3N (2.0 mmol) were stirred in 5 mL of THF
at 60 °C for 12 h. A saturated NaHCO3 (aq) solution (15 mL) was then
added to the crude reaction mixture and stirred at room temp for 1 h.
The reaction mixture was extracted with CH2Cl2 (3 × 20 mL) to afford
the crude product. The residue was loaded onto a Biotage SNAP flash
purification cartridge, eluting with 10% 7 N NH3 in MeOH solution/
CH2Cl2 to give the target compounds 9a−f, 11a-b, and 14. Compounds
were analyzed for purity using LCMS, 1H and 13C NMR spectroscopy,
and, if necessary, purified further using a Biotage SNAP flash pur-
ification cartridge, eluting with a 10% 7 N NH3 in MeOH solution/
EtOAc. It should be noted; the following yields of the target compounds
were not optimized.
(d, JC–C–F = 18.2 Hz), 125.2 (2xC), 116.3 (2xC) (d, J(1)C–C–F = 22.0 Hz;
J
J
(1)C–C–F = 22.0 Hz), 83.7 (d, JC–F = 169.5 Hz, CH2F), 83.6 (d,
C–F = 169.7 Hz, CH2F), 78.5, 77.6, 68.2 (d, JC–F = 19.6 Hz,
O–CH2CH2F), 52.8 (2xC), 51.2, 45.7 (2xC), 44.0, 37.8, 31.6, 29.9; LC-
MS (ESI) m/z: 414.19 [M+H].
4.1.3.4. (S)-4-(4-fluoro-3-(3-(2-fluoroethoxy)pyrrolidine-1-carbonyl)
benzyl)phthalazin-1(2H)-one (9d). Compound 9d can be prepared
following the general synthetic procedure to afford a white crystalline
solid (Yield 14%). 1H NMR (500 MHz, CDCl3) (reported as mixture of
rotamers) δ 11.65 (s, 1H), 8.45–8.44 (m, 1H), 7.72–7.71 (m, 3H),
7.39–7.36 (m, 1H), 7.28–7.26 (m, 1H), 7.00–6.96 (m, 1H), 4.57–4.40
(m, 2H), 4.27 (s, 2H), 4.19–4.08 (m, 1H), 3.77–3.71 (m, 2H), 3.69–3.64
(m, 1H), 3.59–3.42 (m, 2H), 3.32–3.28 (m, 1H), 2.09–1.93 (m, 2H); 13
C
4.1.3.1. 4-(4-fluoro-3-(6-(2-fluoroethoxy)-2-azaspiro[3.3]heptane-2-
carbonyl)benzyl)phthalazin-1(2H)-one (9a). Compound 9a can be
prepared following the general synthetic procedure to afford a white
crystalline solid (Yield 28%). 1H NMR (500 MHz, CDCl3) (reported as
mixture of rotamers) δ 11.23 (m, 1H), 8.47–8.45 (m, 1H), 7.77–7.73 (m,
2H), 7.72–7.69 (m, 1H), 7.49–7.46 (m, 1H), 7.31–7.28 (m, 1H),
7.00–6.96 (m, 1H), 4.54–4.53 (t, J = 4.1 Hz, 1H), 4.45–4.43 (t,
J = 4.0 Hz, 1H), 4.27 (s, 2H), 4.13 (s, 1H), 3.98 (s, 1H), 3.96–3.83
(m, 1H), 3.59–3.56 (m, 1H), 3.53–3.50 (m, 1H), 2.56–2.52 (m, 1H),
2.50–2.46 (m, 1H), 2.21–2.17 (m, 1H), 2.14–2.10 (m, 1H); 13C NMR
(125 MHz, CDCl3) (reported as mixture of rotamers) δ 166.0 (2xC), 160.8,
157.0 (d, JC–F = 250.0 Hz), 156.9 (d, JC–F = 249.0 Hz), 145.7, 134.1,
(d, JC–C–C–C–F = 3.2 Hz), 133.7, 132.3 (d, JC–C–C–F = 7.2 Hz), 130.8 (d,
JC–C–C–F = 7.6 Hz), 131.6, 130.2 (d, JC–C–C–F = 3.4 Hz), 130.1 (d,
NMR (125 MHz, CDCl3) (reported as mixture of rotamers) δ 165.1 (2xC),
161.0, 156.3 (2xC) (d, J(1)C–F = 247.6 Hz; J(2)C–F = 247.6 Hz), 145.7,
134.2 (d, JC–C–C–C–F = 3.2 Hz), 134.1 (d, JC–C–C–C–F = 3.0 Hz), 133.7,
131.5, 131.4 (2xC) (d, J(1)C–C–C–F = 8.1 Hz; J(2)C–C–C–F = 7.4 Hz), 129.6,
129.1 (d, JC–C–C–F = 3.8 Hz), 128.9 (d, JC–C–C–F = 3.7 Hz), 128.3, 127.1,
125.5 (d, JC–C–F = 18.5 Hz), 125.4 (d, JC–C–F = 18.3 Hz), 125.2 (2xC),
116.4 (2xC) (d, J(1)C–C–F = 22.0 Hz; J(1)C–C–F = 22.0 Hz), 83.7 (d,
J
C–F = 169.3 Hz, CH2F), 83.6 (d, JC–F = 169.8 Hz, CH2F), 78.5, 77.6,
68.2 (d, JC–F = 19.9 Hz, O–CH2CH2F), 52.8 (2xC), 51.2, 45.8 (2xC),
44.0, 37.8, 31.6, 29.9; LC-MS (ESI) m/z: 414.19 [M+H].
4.1.3.5. 4-(4-fluoro-3-(4-(2-fluoroethoxy)piperidine-1-carbonyl)benzyl)
phthalazin-1(2H)-one (9e). Compound 9e can be prepared following
the general synthetic procedure to afford a white crystalline solid (Yield
37%). 1H NMR (500 MHz, CDCl3) δ 11.84 (s, 1H), 8.45–8.43 (m, 1H),
7.73–7.67 (m, 3H), 7.30–7.28 (m, 1H), 7.27–7.24 (m, 1H), 6.98–6.95 (t,
J = 8.8 Hz, 1H), 4.56–4.54 (t, J = 4.1 Hz, 1H), 4.46–4.44 (t,
J = 4.1 Hz, 1H), 4.26 (s, 2H), 3.96 (bs, 1H), 3.73–3.68 (m, 1H),
3.67–3.63 (m, 1H), 3.62–3.60 (m, 1H), 3.55 (bs, 1H), 3.43 (bs, 1H),
J
C–C–C–F = 3.5 Hz), 129.6, 128.4, 127.2, 125.2, 122.5 (d,
JC–C–F = 17.6 Hz),
122.4
116.5
(d,
(d,
JC–C–F = 17.0 Hz),
JC–C–F = 22.7 Hz),
116.6
83.7
(d,
(d,
J
J
C–C–F = 22.5 Hz),
C–F = 169.4 Hz, CH2F), 83.6 (d, JC–F = 169.4 Hz, CH2F), 68.9, 68.8,
67.5 (d,
JC–F = 19.7 Hz, O–CH2CH2F), 67.4 (d, JC–F = 19.7 Hz,
O–CH2CH2F), 63.5, 63.4, 62.3, 62.2, 60.8, 59.7, 41.0, 37.8 (2xC),
30.7, 30.6; LC-MS (ESI) m/z: 440.09 [M+H].
3.08 (bs, 1H), 1.91–1.87 (m, 1H), 1.72–1.66 (m, 2H), 1.55 (bs, 1H); 13
C
NMR (125 MHz, CDCl3) δ 164.8, 161.0, 156.1 (d, JC–F = 247.2 Hz),
145.6, 134.2 (d, JC–C–C–C–F = 3.1 Hz), 133.6, 131.5, 131.1 (d,
4.1.3.2. 4-(4-fluoro-3-(3-(2-fluoroethoxy)azetidine-1-carbonyl)benzyl)
phthalazin-1(2H)-one (9b). Compound 9b can be prepared following
the general synthetic procedure to afford a white crystalline solid (Yield
10%). 1H NMR (500 MHz, CDCl3) δ 11.42 (s, 1H), 8.47–8.45 (m, 1H),
7.77–7.70 (m, 3H), 7.51–7.50 (dd, J1 = 1.9 Hz, J2 = 4.2 Hz, 1H),
7.32–7.29 (m, 1H), 6.95 (t, J = 9.1 Hz, 1H), 4.58–4.56 (t, J = 4.0 Hz,
1H), 4.49–4.47 (t. J = 4.0 Hz, 1H), 4.38–4.33 (m, 2H), 4.27 (s, 2H),
4.19–4.16 (m, 1H), 4.07–4.06 (m, 1H), 4.00–3.98 (m, 1H), 3.71–3.56
(m, 2H); 13C NMR (125 MHz, CDCl3) δ 166.2, 160.9, 156.9 (d,
JC–F = 250.4 Hz), 145.7, 134.1 (d, JC–C–C–C–F = 3.4 Hz), 133.7, 132.4
JC–C–C–F = 7.9 Hz), 129.5, 128.9 (d, JC–C–C–F = 3.7 Hz), 128.2, 127.0,
125.1, 124.5 (d, JC–C–F = 18.7 Hz), 116.1 (d, JC–C–F = 21.6 Hz), 83.8 (d,
J
C–F = 169.4 Hz, CH2F), 74.2, 68.2 (d, JC–F = 19.3 Hz, O–CH2CH2F),
44.1, 38.8, 37.7, 31.3, 30.3; LC-MS (ESI) m/z: 428.08 [M+H].
4.1.3.6. 4-(4-fluoro-3-(4-(2-fluoroethoxy)azepane-1-carbonyl)benzyl)
phthalazin-1(2H)-one (9f). Compound 9f can be prepared following the
general synthetic procedure to afford a white crystalline solid (Yield
41%). 1H NMR (500 MHz, CDCl3) (reported as mixture of isomers and
rotamers) δ 11.76–11.74 (m, 1H), 8.45–8.43 (m, 1H), 7.73–7.69 (m,
3H), 7.28–7.23 (m, 2H), 7.28–7.24 (m, 2H), 6.99–6.95 (m, 1H),
4.56–4.53 (m, 1H), 4.46–4.42 (m, 1H), 4.26 (s, 2H), 3.74 (bs, 1H),
3.69–3.65 (m, 1H), 3.62–3.56 (m, 2H), 3.55–3.51 (m, 1H), 3.39–3.20
(m, 2H), 2.04–1.91 (m, 2H), 1.87–1.77 (m, 2H), 1.74–1.68 (m, 2H),
1.55 (bs, 1H); 13C NMR (125 MHz, CDCl3) (reported as mixture of isomers
and rotamers) δ 166.5, 166.3, 161.0 (2xC), 155.9 (d, JC–F = 247.4 Hz),
145.7, 134.2 (d, JC–C–C–C–F = 3.5 Hz), 134.1 (d, JC–C–C–C–F = 3.5 Hz),
133.6 (2xC), 131.5, 130.9 (d, JC–C–C–F = 7.5 Hz), 130.8 (d,
(d,
JC–C–C–F = 8.3 Hz), 131.6, 130.2 (d, JC–C–C–F = 3.4 Hz), 129.6,
128.3, 127.2, 125.0, 122.2 (d, JC–C–F = 16.9 Hz), 116.4 (d,
J
J
C–C–F = 22.7 Hz), 82.2 (d, JC–F = 169.9 Hz, CH2F), 68.6, 68.5 (d,
C–F = 19.7 Hz, O–CH2CH2F), 58.5, 58.4, 55.9, 37.8; LC-MS (ESI) m/
z: 400.17 [M+H].
4.1.3.3. (R)-4-(4-fluoro-3-(3-(2-fluoroethoxy)pyrrolidine-1-carbonyl)
benzyl)phthalazin-1(2H)-one (9c). Compound 9c can be prepared
following the general synthetic procedure to afford a white crystalline
solid (Yield 12%). 1H NMR (500 MHz, CDCl3) (reported as mixture of
rotamers) δ 11.81 (s, 1H), 8.45–8.43 (m, 1H), 7.74–7.70 (m, 3H),
7.38–7.35 (m, 1H), 7.27–7.25 (m, 1H), 6.99–6.96 (m, 1H), 4.57–4.39
(m, 2H), 4.26 (s, 2H), 4.19–4.06 (m, 1H), 3.76–3.70 (m, 2H), 3.69–3.65
(m, 1H), 3.63–3.41 (m, 2H), 3.32–3.27 (m, 1H); 2.08–2.02 (m, 1H),
2.01–1.92 (m, 1H); 13C NMR (125 MHz, CDCl3) (reported as mixture of
J
C–C–C–F = 7.6 Hz), 129.6, 128.5 (d, JC–C–C–F = 4.0 Hz), 128.2, 127.1,
125.5 (d, JC–C–F = 19.0 Hz), 125.4 (d, JC–C–F = 19.0 Hz), 116.3 (d,
JC–C–F = 21.7 Hz),
116.2
(d,
JC–C–F = 21.7 Hz),
84.0
(d,
J
C–F = 169.2 Hz, CH2F), 83.9 (d, JC–F = 168.9 Hz, CH2F), 77.5, 77.2,
67.7 (d,
JC–F = 19.7 Hz, O–CH2CH2F), 67.6 (d, JC–F = 20.0 Hz,
O–CH2CH2F), 48.7, 45.9, 43.8, 40.6, 37.8, 34.4, 32.9, 31.8, 30.1,
21.8, 21.3; LC-MS (ESI) m/z: 442.25 [M+H].
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BioorganicChemistry83(2019)242–249
4.1.3.7. 4-(4-fluoro-3-(4-(4-fluorophenyl)piperazine-1-carbonyl)benzyl)
phthalazin-1(2H)-one (11a). Compound 11a can be prepared following
the general synthetic procedure to afford a white crystalline solid (Yield
52%). 1H NMR (500 MHz, ((CD3)2SO) δ 12.60 (s, 1H), 8.27 (d,
J = 7.7 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.2 Hz, 1H),
7.82 (t, J = 7.5 Hz, 1H), 7.45–7.43 (m, 1H), 7.38–7.37 (m, 1H), 7.24 (t,
J = 9.1 Hz, 1H), 7.06 (t, J = 8.6 Hz, 2H), 6.96–6.93 (m, 2H), 4.33 (s,
2H), 3.75 (bs, 2H), 3.30 (bs, 2H), 3.12 (bs, 2H), 2.95 (bs, 2H). 13C NMR
purged with N2 for 5 min, followed by the addition of 15 (2.0 mmol)
in 2.5 mL of 2-methyl-2-butanol. The reaction mixture was heated to
80 °C and stirred vigorously for 12 h. After which, the crude reaction
mixture was filtered and the solvent was removed under reduced
pressure. The subsequent residue was loaded onto a Biotage SNAP
flash purification cartridge, eluting with a 1:5 EtOAc/hexanes gradient,
to afford the Boc-protected precursor of 16 as a white solid. (Yield
74%). The intermediate was then dissolved in CH2Cl2 (2 mL), followed
by dropwise addition of CF3COOH (2 mL), and stirred at room
temperature for 3 h. Volatiles were then removed under reduced
pressure and the crude product was neutralized with a saturated
NaHCO3 (aq) solution (10 mL). The reaction mixture was extracted
with CH2Cl2 (3 × 20 mL), and the organic layers were combined, dried,
and concentrated to afford the free-amine intermediates (16) as light-
(125 MHz, ((CD3)2SO))
δ
163.8, 159.3, 155.4 (2xC) (d,
1C–F = 240.6 Hz, J2C–F = 239.1 Hz), 147.5, 144.8, 134.8 (d,
C–C–C–C–F = 3.8 Hz), 133.4, 131.6 (d, JC–C–C–F = 7.9 Hz), 131.5,
J
J
129.0, 128.8 (d, JC–C–C–F = 3.9 Hz), 127.9, 126.0, 125.4, 123.6 (d,
J
J
C–C–F = 18.3 Hz),
117.9
(d,
JC–C–C–F = 7.8 Hz),
116.0
(d,
C–C–F = 21.8 Hz), 115.4 (d, JC–C–F = 21.9 Hz), 49.3, 49.2, 46.3, 41.2,
36.4; LC-MS (ESI) m/z: 461.06 [M+H].
tan solid. Finally, 16 (1.0 mmol), 8 (1.0 mmol), HOBt hydrate
(1.0 mmol), EDC hydrochloride (1.0 mmol), and Et3N (2.0 mmol)
were stirred in 5 mL of THF at 60 °C for 12 h. A saturated NaHCO3
(aq) solution (15 mL) was then added to the crude reaction mixture and
stirred at room temp for 1 h. The reaction mixture was extracted with
CH2Cl2 (3 × 20 mL) to afford the crude product. The residue was
loaded onto a Biotage SNAP flash purification cartridge, eluting with
10% 7 N NH3 in MeOH solution/CH2Cl2 to give the target compounds
17 as a soft white solid. (Yield 18%) 1H NMR (500 MHz, CDCl3)
(reported as mixture of rotamers) δ 11.38 (m, 1H), 8.48–8.46 (m, 1H),
7.77–7.71 (m, 3H), 7.52–7.49 (m, 1H), 7.34–7.29 (m, 1H), 7.10–7.06
(m, 2H), 7.01–6.93 (m, 3H), 4.29–4.28 (m, 3H), 4.16 (s, 1H), 4.08 (s,
1H), 3.94 (s, 1H), 3.42–3.25 (m, 1H), 2.64–2.60 (m, 1H), 2.58–2.54 (m,
1H), 2.31–2.27 (m, 1H), 2.24–2.19 (m, 1H). 13C NMR (125 MHz, CDCl3)
(reported as mixture of rotamers) δ 166.1, 166.0, 160.9, 160.4 (2xC) (d,
4.1.3.8. 4-(4-fluoro-3-(4-(4-fluorophenyl)piperidine-1-carbonyl)benzyl)
phthalazin-1(2H)-one (11b). Compound 11b can be prepared following
the general synthetic procedure to afford a hard white solid (Yield
28%). 1H NMR (500 MHz, ((CD3)2SO) δ 12.60 (s, 1H), 8.25 (d,
J = 7.6 Hz, 1H), 7.85–7.82 (m, 1H), 7.79–7.76 (m, 1H), 7.42–7.40
(m, 2H), 7.25–7.19 (m, 3H), 7.10 (t, J = 8.9 Hz, 1H), 4.64–4.61 (m,
1H), 4.33 (s, 2H), 3.40–3.38 (m, 1H), 3.10 (t, J = 12.4 Hz, 1H),
2.83–2.75 (m, 2H), 1.83–1.81 (m, 1H), 1.63–1.49 (m, 2H), 1.40 (bs,
1H). 13C NMR (125 MHz, ((CD3)2SO))
δ
163.6, 159.7 (d,
JC–F = 243.0 Hz), 159.3, 155.3 (JC–F = 243.0 Hz), 144.8, 141.4 (d,
J
C–C–C–C–F = 2.6 Hz), 134.7 (d, JC–C–C–C–F = 2.9 Hz), 133.3, 131.4,
131.3 (d, C–C–C–F = 7.8 Hz), 129.0, 128.5, 128.4 (d,
J
J
C–C–C–F = 7.8 Hz), 127.9, 126.0, 125.4, 124.3 (d, JC–C–F = 18.2 Hz),
115.9 (d, JC–C–F = 22.0 Hz), 114.9 (d, JC–C–F = 20.7 Hz), 59.7, 54.86,
46.9, 41.6, 40.7, 36.4, 32.6; LC-MS (ESI) m/z: 460.12 [M+H].
J1C–F = 244.7 Hz, J2C–F = 244.7 Hz), 157.0 (JC–F = 250.4 Hz), 145.7,
140.0 (d, JC–C–C–C–F = 2.8 Hz), 139.9 (d, JC–C–C–C–F = 2.7 Hz), 134.1,
134.0 (d, JC–C–C–F = 8.1 Hz), 133.7, 132.2 (d, JC–C–C–F = 8.2 Hz), 131.6,
4.1.3.9. 4-(4-fluoro-3-(6-(4-fluorophenyl)-2,6-diazaspiro[3.3]heptane-2-
carbonyl)benzyl)phthalazin-1(2H)-one (14). Compound 14 can be
prepared following the general synthetic procedure to afford a hard
white solid (Yield 26%). 1H NMR (500 MHz, CDCl3) δ 11.83 (s, 1H),
8.25 (d, J = 7.7 Hz, 1H), 7.75–7.70 (m, 3H), 7.52–7.50 (dd,
J1 = 2.0 Hz, J2 = 6.2 Hz, 1H), 7.34–7.31 (m, 1H), 7.00 (t, J = 9.2 Hz,
1H), 6.87 (t, J = 8.7 Hz, 1H), 6.34–6.32 (m, 2H), 4.31 (s, 2H), 4.28 (s,
130.2, 129.6, 128.4, 127.7 (d, JC–C–C–C–F = 3.2 Hz), 127.6 (d,
J
C–C–C–C–F = 2.8 Hz), 127.2, 125.2, 122.4 (d, JC–C–F = 17.2 Hz), 122.3
(d, JC–C–F = 17.2 Hz), 116.3 (d, JC–C–F = 22.7 Hz), 115.1 (d,
J
C–C–F = 21.2 Hz), 64.0, 63.9, 61.9, 61.8, 61.4, 59.3, 40.4, 37.8, 34.4,
34.2, 33.6, 33.3; LC-MS (ESI) m/z: 472.14 [M+H].
4.1.5. Synthetic procedure for compound 19a-b
2H), 4.20 (s, 2H), 3.94 (d, J = 7.6 Hz, 2H), 3.89 (d, J = 7.6 Hz, 2H). 13
C
4.1.5.1. 4-(4-fluoro-3-(4-(2-hydroxyethoxy)piperidine-1-carbonyl)benzyl)
phthalazin-1(2H)-one (15). Compounds 8 (1.0 mmol), 2-(piperidin-4-
yloxy)ethan-1-ol (1. 0 mmol), HOBt hydrate (1.0 mmol), EDC
hydrochloride (1.0 mmol), and Et3N (2.0 mmol) were stirred in 5 mL
of THF at 60 °C for 12 h. A saturated NaHCO3 (aq) solution (15 mL) was
then added to the crude reaction mixture and stirred at room temp for
1 h. The reaction mixture was extracted with CH2Cl2 (3 × 20 mL) to
afford the crude product. The residue was loaded onto a Biotage SNAP
flash purification cartridge, eluting with 10% 7 N NH3 in MeOH
NMR (125 MHz, CDCl3) δ 166.6, 161.1, 156.9 (d, JC–F = 250.2 Hz),
155.4 (JC–F = 236.0 Hz), 147.6, 145.6, 134.2 (d, JC–C–C–C–F = 3.0 Hz),
133.7,
132.6
(d,
J
C–C–C–F = 8.3 Hz),
131.5,
130.2
115.6
(d,
(d,
J
J
J
C–C–C–C–F = 3.3 Hz), 129.6, 128.2, 127.1, 125.1, 122.0 (d,
C–C–F = 16.8 Hz),
116.3
(d,
JC–C–F = 22.5 Hz),
C–C–F = 22.3 Hz), 112.8 (d, JC–C–C–F = 7.5 Hz), 62.5, 61.2, 61.1, 58.6,
37.7, 33.7; LC-MS (ESI) m/z: 473.21 [M+H].
4.1.4. Synthetic procedure for compound 17
solution/CH2Cl2 to give the target compounds 15 as a white
4.1.4.1. 4-(4-fluoro-3-(6-(4-fluorophenyl)-2-azaspiro[3.3]heptane-2-
carbonyl)benzyl)phthalazin-1(2H)-one (17). The synthesis of 16 was
adapted from previously reported literature conditions [16,24,25]. A
solution of 5a (6.0 mmol) in THF (20 mL) at 0 °C was treated with
triphenylphosphine (10 mmol), followed by CBr4 (10 mmol). After 1 h,
the reaction mixture was warmed to room temp, and stirred for an
additional 2 h. Next, the solvent was removed under reduced pressure
to afford a crude oily residue. The subsequent residue was loaded onto a
Biotage SNAP flash purification cartridge, eluting with a 1:20 EtOAc/
hexanes gradient, to afford 15 as a white solid. (Yield 26%) 1H NMR
(500 MHz, CDCl3) δ 4.27–4.24 (quint, J = 7.3 Hz, 1H), 3.89 (s, 2H),
3.86 (s, 2H), 2.83–2.79 (m, 2H), 2.57–2.52 (m, 2H), 1.37 (s, 9H). 13C
NMR (125 MHz, CDCl3) δ 156.0, 79.4, 61.0, 60.7, 45.6, 36.4, 35.6,
28.3; LC-MS (ESI) m/z: 176.18 [M-Boc]. A 40 mL vial was charged with
crystalline solid. (Yield 38%) 1H NMR (500 MHz, CDCl3) δ 11.78 (s,
1H), 8.42–8.40 (m, 1H), 7.69–7.67 (m, 3H), 7.28–7.26 (m, 1H),
7.25–7.26 (m, 1H), 6.97–6.93 (m, 1H), 4.24 (m, 2H), 4.01 (bs, 1H),
3.69–3.68 (m, 2H), 3.57–3.50 (m, 3H), 3.43 (bs, 2H), 3.05 (bs, 1H),
2.81 (bs, 1H), 1.91–1.88 (m, 1H), 1.73 (bs, 1H), 1.68–1.62 (m, 1H),
1.52 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 164.8, 160.9, 156.0 (d,
J
C–F = 246.9 Hz), 145.7, 134.2 (d, JC–C–C–C–F = 3.1 Hz), 133.6, 131.5,
131.1 (d, JC–C–C–F = 7.8 Hz), 129.5, 128.9 (d, JC–C–C–F = 3.5 Hz), 128.2,
127.0, 125.1, 124.5 (d, JC–C–F = 18.6 Hz), 116.1 (d, JC–C–F = 22.0 Hz),
74.2, 69.4, 61.8, 44.3, 39.0, 37.7, 31.3, 30.5; LC-MS (ESI) m/z: 426.06
[M+H].
4.1.5.2. 2-((1-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)
benzoyl)piperidin-4-yl)oxy)ethyl 4-methylbenzenesulfonate (19a). Reagent
p-toulenesulfonyl chloride (1.3 mmol) was slowly added to a stirring
solution of 18 (1.0 mmol) and TEA (2.1 mmol) in 20 mL of DCM at 0 °C.
The reaction mixture was allowed to warm to room temp, and stirred for
NiI2
(0.20 mmol),
trans-2-aminocyclohexanol
hydrochloride
(0.20 mmol), (4-fluorophenyl)boronic acid (2.0 mmol), NaHMDS
(4.0 mmol), and 2-methyl-2-butanol (1.0 mL). The reaction was
247

S.W. Reilly et al.
BioorganicChemistry83(2019)242–249
12 h. Then, the reaction mixture was filtered and solvent was removed
under reduced pressure to afford a crude oily reside. The subsequent
residue was loaded onto a Biotage SNAP flash purification cartridge,
eluting with a 1:20 MeOH/EtOAc gradient, to afford 19a as a white
crystalline solid. (Yield 75%) 1H NMR (500 MHz, CDCl3) δ 11.61 (s, 1H),
8.45–8.43 (m, 1H), 7.76–7.74 (m, 3H), 7.73–7.69 (m, 2H), 7.31–7.29 (m,
3H), 7.28–7.25 (m, 1H), 6.99–6.96 (t, J = 8.7 Hz, 1H), 4.26 (s, 2H),
4.13–4.11 (t, J = 4.7 Hz, 2H), 3.79 (bs, 1H), 3.66–3.58 (m, 3H), 3.52 (bs,
1H), 3.35 (bs, 1H), 3.05 (bs, 1H), 2.40 (s, 3H), 1.80–1.76 (m, 1H), 1.65
(bs, 1H), 1.58–1.56 (m, 1H), 1.47 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ
164.8, 160.9, 156.0 (d, JC–F = 247.6 Hz), 145.7, 144.9, 134.2 (d,
JC–C–C–C–F = 3.3 Hz), 133.6, 133.0, 131.5, 131.2 (d, JC–C–C–F = 7.9 Hz),
129.8, 129.6, 128.9 (d, JC–C–C–F = 3.4 Hz), 128.3, 127.9, 127.1, 124.5,
125.1, 124.5 (d, JC–C–F = 18.4 Hz), 116.1 (d, JC–C–F = 22.0 Hz), 74.1,
69.5, 65.7, 43.9, 38.6, 37.7, 31.3, 30.5, 21.6; LC-MS (ESI) m/z: 426.06
[M+H]; LC-MS (ESI) m/z: 580.20 [M+H].
incubation, 50 µl of ATP Detection Reagent was added to all wells to
stop the P-gp reaction. Samples were mixed briefly on a plate shaker
then incubated plate at room temperature for 20 min to allow lumi-
nescent signal to develop. Luminescence was read on a plate-reading
luminometer. This luciferase-based detection reaction provides a linear
response to ATP concentration in each sample. Thus any changes in
signal directly reflect changes in ATP concentration.
4.2.3. Radiochemistry
The radiosynthesis of [18F]9e was accomplished on a Synthera
synthesis module with full automation. Briefly, [18F]fluoride (800 mCi)
was produced by proton irradiation of enriched H218O via the reaction
of 18O (p, n) 18F by the Cyclone cyclotron (IBA). The [18F]fluoride in a
H218O solution is delivered to the hotcell, trapped on a preactivated
Sep-Pak Light QMA Carb cartridge (Waters), and eluted to the reaction
vial with 1 mL of eluent containing 2 mg of potassium carbonate and
7 mg of Kryptofix in a mixture of 0.85 mL of acetonitrile and 0.15 mL of
water. The residual water was evaporated azotropically with 1 mL of
acetonitrile at 100 °C under a stream of nitrogen gas and vacuum. A
solution of 4 mg of tosylate precursor 19a in 0.7 mL of methyl sulfide
(DMSO) was added to the reaction vial for a 20 min reaction. The crude
product was diluted with 3 mL of the mobile phase and passed through
an Alumina N Light cartridge (Waters) and a 0.45 μm nylon filter to the
4.1.5.3. 2-((1-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)
benzoyl)piperidin-4-yl)oxy)ethyl
methanesulfonate
(19b). Reagent
methane sulfonyl chloride (1.3 mmol) was slowly added to a stirring
solution of 18 (1.0 mmol) and TEA (2.1 mmol) in 20 mL of DCM at 0 °C.
The reaction mixture was allowed to warm to room temp, and stirred
for 12 h. Then, the reaction mixture was filtered and solvent was
removed under reduced pressure to afford a crude oily reside. The
subsequent residue was loaded onto a Biotage SNAP flash purification
cartridge, eluting with a 1:20 MeOH/EtOAc gradient, to afford 19b as a
white crystalline solid. (Yield 80%) 1H NMR (500 MHz, CDCl3) δ 11.33
(s, 1H), 8.46–8.44 (m, 1H), 7.75–7.71 (m, 3H), 7.30–7.26 (m, 2H),
7.01–6.97 (t, J = 8.8 Hz, 1H), 4.34–4.33 (t, J = 4.5 Hz, 2H), 4.27 (s,
2H), 3.98 (bs, 1H), 3.76–3.68 (m, 2H), 3.61 (bs, 1H), 3.53 (bs, 1H),
3.45–3.43 (m, 1H), 3.11 (bs, 1H), 3.03 (s, 3H), 1.92–1.88 (m, 1H), 1.75
(bs, 1H), 1.71–1.66 (m, 1H), 1.54 (bs, 1H); 13C NMR (125 MHz, CDCl3)
HPLC loop for high-performance chromatography (HPLC).
A
Phenomenex Luna 5 µm C18 100 Å LC Column 250 × 10 mm semi-
preparative column with a mobile phase of acetonitrile and water
(40:60 by volume) was used for HPLC purification. At a flow rate of
4 mL/min, the product was eluted at 11 min and diluted with water to a
volume of 50 mL. The diluted product solution was passed through a
Sep-Pak Plus C18 cartridge (Waters). The trapped product was rinsed
with water to waste and then eluted with ethanol (0.6 mL, with 0.1%
ascorbic acid) followed by 8 mL of normal saline to the final production
vial through a 0.2 μm nylon filter. After being shaken well, the final
product was ready for quality control (QC) and animal studies. The
yield ranged from 8 to 11% (decay corrected to the start of synthesis) in
an average time of 65 min from receipt of [18F]fluoride in a H218O
solution from the cyclotron.
δ
164.9, 160.8, 156.1 (d, JC–F = 246.7 Hz), 145.7, 134.2 (d,
J
C–C–C–C–F = 3.2 Hz),
133.7,
133.0,
131.6,
131.2
(d,
JC–C–C–F = 7.7 Hz), 129.6, 129.0 (d, JC–C–C–F = 3.7 Hz), 128.3, 127.1,
125.1, 124.6 (d, JC–C–F = 18.5 Hz), 116.2 (d, JC–C─F = 21.8 Hz), 74.5,
69.3, 66.0, 44.1, 38.9, 37.8, 37.7, 31.3, 30.4; LC-MS (ESI) m/z: 504.86
[M+H].
4.2.4. In vitro autoradiography
The normal Balb/c mouse (male, 10–12 weeks-old) frozen brain
sections (10-μm thickness) were prepared using a cryostat microtome
(CM1900, Leica, Germany) one day before and kept in -80 °C freezer.
The sections were first thawed at room temperature for 20 min and
rehydrated with ice-cold PBS buffer (pH 7.4) for 5 min. Then all sec-
tions were incubated with 4 nM [18F]9e with, or without, 10 μM ola-
parib at room temperature for 1 h to define the control and nonspecific
binding groups. After the radioligand incubation, all of them were
washed three times with ice-cold PBS for 3 min, dried up with a fan, and
exposed to a BAS-SR 2040 imaging plate (20 × 40 cm, Fujifilm, Japan)
for 2 hr. In the end the plate were scanned with a Typhoon 7000
phosphorimager (GE Healthcare) in a condition of 500 V and a re-
solution of 25 μm.
4.2. Biological evaluation
4.2.1. PARP-1 radioligand binding
The affinity of each compound for the PARP-1 enzyme was eval-
uated using a radioligand binding method previously reported by our
group [17]. Briefly, OVCAR8 ovarian cancer cells were seeded in 96-
well Stripwell plates at a density of 40,000 cells/well 24 hrs prior to the
study. On the day of study, compounds were diluted in RPMI to con-
centrations of 100 µM - 0.064 nM. Next, [125I]KX1, a known PARP-1
specific radioligand, was added to the plate followed by each compound
dilution. Reactions were allowed to equilibrate for 1 hr and were then
washed with 200 µl of PBS. Wells were separated and counted on an
automatic Wizard gamma counter (Perkin Elmer, Waltham MA). Dose
response curves were produced to calculate 50% maximum inhibition
values (IC50) using non-linear fit sigmoidal dose response curves in
GraphPad 7.0 (Prism, La Jolla CA). Experiments were repeated three
times with adjusting dose concentrations to increase accuracy of IC50
values.
4.2.5. MicroPET imaging
All animal studies were performed under protocols approved by the
University of Pennsylvania Institutional Animal Care and Use
Committee. Male rhesus macaques (13–21 year old) were sedated with
ketamine/dexdomidor and anesthesia maintained for imaging with 1%
isoflurane. Temperature was maintained with a recirculating water
warm pad and vital signs such as blood pressure, pulse oximetry, and
EKG were monitored continuously. Three rhesus macaques were
scanned on the G-PET, a high sensitivity, high resolution PET scanner
using gadolinium orthosilicate crystals incorporated into an Anger-logic
detector developed for brain imaging. Data was acquired for up to
50 min (6 × 10 sec, 3 × 60 sec, 3 × 120 sec, 3 × 180 sec, 4 × 300 sec,
and 1 × 600 sec) in list mode after an intravenous injection of
4.2.2. Pgp-Glo™ assay
P-gp activity of each compound was measured using Promega Pgp-
Glo™ Assay Systems. 25 µg of diluted recombinant human P-gp mem-
branes were added to untreated white opaque multiwell plates along
with Pgp-Glo™ Assay Buffer, a non-limiting concentration of ATP
(5 mM) and 20 µl of each test compound (20 µM) for 1 hr at 37 °C.
Untreated and Na3VO4-treated control samples were also tested in
addition to Verapamil-treated samples (positive control). After
248

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BioorganicChemistry83(2019)242–249
101.6 MBq
[
18F]FTT to one of the rhesus macaques, and
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sample was drawn into a heparinized syringe at 37 min post [18F]9
injection for metabolite analysis. The acquired data were sorted into
sinograms and reconstructed using the fully 3D LOR-RAMLA iterative
reconstruction algorithm. Six volumes of interest (VOIs) including
striatum, thalamus, frontal cortex, occipital cortex, whole brain, and
cerebellum were manually delineated with PMOD image analysis soft-
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4.2.6. Metabolite analysis
The blood samples were centrifuged at 3000 G for 10 min to sepa-
rate plasma and red blood cells. 2 mL acetonitrile was added to the
sample of plasma (1 mL). The plasma solution was vortexed, followed
by centrifugation at 3000 G for 10 min. After the supernatant had been
separated from the pellet, each portion was counted with a gamma
counter (PerkinElmer Wizard 2480) to determine the extraction effi-
ciency. Each supernatant was diluted with water and passed through a
0.45 μm nylon filter for HPLC injection. With Agilent 1200 series,
200 μL solution was injected onto Agilent SB-C18 column
(250 × 10 mm) for analysis. The mobile phase was 34% acetonitrile in
water (volume) and the flow rate was 1 mL/min. The HPLC eluent was
collected 1 tube/min and 16 tubes were collected for each injection.
The collected HPLC fractions were measured by Gamma Counter for
further data analysis. Meanwhile the cold reference was also injected
and monitored at 254 nm UV wavelength.
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Funding sources
SWR and LNP conducted this research through the support of
training grants 5T32DA028874-07 and T32GM008076, respectively.
Acknowledgment
[19] J.D. Hughes, J. Blagg, D.A. Price, S. Bailey, G.A. DeCrescenzo, R.V. Devraj,
E. Ellsworth, Y.M. Fobian, M.E. Gibbs, R.W. Gilles, N. Greene, E. Huang, T. Krieger-
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David Livingston (Harvard Medical School) for the OVCAR8 cancer
cells.
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A. Wang-Gillam, C. Bognar, W. Chu, D. Zhou, R.H. Mach, R. Laforest, D.L. Chen,
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first in-human studies, Radiology 282 (2017) 453–463.
Appendix A. Supplementary material
Monkey blood, and mice brain and blood HPLC metabolite analysis
of [18F]9e (PDF). 1H and 13C NMR spectra of compounds 9a-f, 11a-b,
14, 17, and 19a (PDF). Supplementary data to this article can be found
online at https://doi.org/10.1016/j.bioorg.2018.10.015.
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drugs, Chem. Rev. 109 (2009) 2989–3011.
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K.J. Dillon, J. Drzewiecki, S. Garman, S. Gomez, H. Javaid, F. Kerrigan, C. Knights,
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