Synthesis, [18F] radiolabeling, and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography

Article abstract of DOI:10.1016/j.bmc.2014.01.019

Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several derivatives of benzimidazole carboxamide as PARP-1 inhibitors, which can be ¹⁸F-labeled easily for positron emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition potency for PARP-1 (IC₅₀ = 6.3 nM). [ ¹⁸F]12 was synthesized under conventional conditions in high specific activity with 40-50% decay-corrected yield. MicroPET studies using [ ¹⁸F]12 in MDA-MB-436 tumor-bearing mice demonstrated accumulation of [¹⁸F]12 in the tumor that was blocked by olaparib, suggesting that the uptake of [¹⁸F]12 in the tumor is specific to PARP-1 expression.

Full text of DOI:10.1016/j.bmc.2014.01.019

Bioorganic & Medicinal Chemistry 22 (2014) 1700–1707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, [18F] radiolabeling, and evaluation of poly (ADP-ribose)
polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1
using positron emission tomography
Dong Zhou, Wenhua Chu, Jinbin Xu, Lynne A. Jones, Xin Peng, Shihong Li, Delphine L. Chen,
⇑
Robert H. Mach
Department of Radiology, School of Medicine, Washington University in Saint Louis, St. Louis, MO 63110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for
developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several deriva-
tives of benzimidazole carboxamide as PARP-1 inhibitors, which can be 18F-labeled easily for positron
emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition
potency for PARP-1 (IC50 = 6.3 nM). [18F]12 was synthesized under conventional conditions in high spe-
cific activity with 40–50% decay-corrected yield. MicroPET studies using [18F]12 in MDA-MB-436 tumor-
bearing mice demonstrated accumulation of [18F]12 in the tumor that was blocked by olaparib, suggest-
ing that the uptake of [18F]12 in the tumor is specific to PARP-1 expression.
Received 3 October 2013
Revised 7 January 2014
Accepted 15 January 2014
Available online 24 January 2014
Keywords:
PARP-1
PET
Radiolabeling
Imaging
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
lethality in cancers that depend on PARP1 activity for survival.9
Therefore,
a number of PARP inhibitors, including olaparib
Poly (ADP-ribose) polymerase-1 (PARP-1) is one of the most
abundant members of the PARP family of nuclear enzymes.1 Its
best characterized functions are sensing DNA damage and facilitat-
ing DNA repair, but PARP-1 also participates in many other DNA-
related cellular processes, such as apoptosis regulation, cell divi-
sion, differentiation, transcriptional regulation, and chromosome
stabilization.2–5 PARP-1, a 113 kD protein, has three unique struc-
tural domains: the N-terminal DNA binding domain with two zinc
fingers that specifically bind to damaged DNA strand breaks;1,6 the
central automodification domain; and the C-terminal catalytic do-
main that sequentially transfers ADP-ribose subunits from nicotin-
amide adenine dinucleotide (NAD+) to protein acceptors.7 Due to
its critical role in DNA repair, PARP-1 has been actively pursued
as a drug target over the past 20 years, with tremendous efforts in-
vested to develop several generations of PARP-1 inhibitors (Fig. 1)
for therapeutic purposes, especially in the area of ischemia–reper-
fusion injury and cancer.5,8 More recently, PARP1 inhibition has
been demonstrated as an effective method for inducing synthetic
(AZD2281, KU-59436), veliparib (ABT-888), rucaparib (PF-
01367338, AG014699), and niraparib (MK4827), are now undergo-
ing evaluation in clinical trials as anticancer drugs.10–14 These PARP
inhibitors effectively inhibit PARP1 activity as well as activity of
other PARP-like enzymes such as PARP2 and tankyrase1.15
Despite the promising results from these clinical trials related
to progression-free survival, however, differences in the ability of
the various PARP inhibitors to suppress tumoral PARP activity can-
not be accurately determined by current assays. Additionally, the
mechanism of action for iniparib, initially developed as a PARP
inhibitor, was more recently discovered to be most likely unrelated
to inhibition of PARP activity.16 Therefore, methods that can quan-
tify tumoral PARP activity noninvasively in vivo would be particu-
larly useful for demonstrating tumor-specific PARP inhibition as
well as assessing the duration of effective PARP inhibition to guide
dosing decisions.
Imaging with positron emission tomography (PET) could be an
effective approach for noninvasively determine PARP activity levels
due to its high sensitivity with reasonable spatial resolution, accu-
rate tracer uptake quantification, and minimal physiological effects
from PET tracers radiolabeled with high specificity and purity.
Abbreviations: PARP, poly (ADP-ribose) polymerase; NAD+, nicotinamide
adenine dinucleotide; PET, positron emission tomography.
[
11C]PJ-34, a PARP-1 targeted tracer, showed some potential in
⇑
imaging PARP-1 expression in an animal model of diabetes.17
Recently,
18F-labeled olaparib/AZD2281 derivative was
Corresponding author. Address: University of Pennsylvania, Chemistry Building,
Room 283, 231 S. 34th St, Philadelphia, PA 19104, USA. Tel.: +1 2157468233.
a
E-mail address: rmach@mail.med.upenn.edu (R.H. Mach).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.019

D. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 1700–1707
1701
Table 1
O
NH2
H
N
Inhibition efficiency of PARP-1 inhibitors
N
N
OH
O
O
N
H
F
N
H
H
N
a
R:
O
NH2
O
NU1085 (2) (Ki = 6 nM)
1
PJ-34 ( ) (IC50 = 20 nM)
N
b
c
N
N
O
OR
OR
H
N
N
H
NH
O
N
N
F
N
N
O
A
B
N
N
N
N
Compound
Structure
R
IC50 (nM)
N
F
AZD2281, olaparib (4) (IC50 = 5 nM)
Figure 1. Examples of PARP-1 inhibitors.
1 PJ34
8
/
/
170.2 8.3a
10.8 0.4
25.8 3.3
30.3 5.6
6.3 1.3
O
A
A
A
B
B
B
a
b
c
a
b
c
AG014361 (3) (Ki = 5.8 nM)
9
10
12
13
15
18.7 2.7
22.1 6.3
a
Reported values: IC50 = 20 nM, EC50 = 35 nm.3,24
O
OCH3
NH2
NH
O
OCH3
N
O
OCH3
NH2
NH2
(a)
R
O
+
R
O
N
H
H
H
N
O
N
O
O
5
18F
N
N
OMs
6a
7a
6
7
R = CH2CH2F
R = CH2C CH
R = CH2CH2F
R = CH2C CH
N
N
(a)
O
18F]12
18F]KF, K2222
O
O
OCH3
O
NH2
N
[
16
N
R
O
(b)
R
O
(c)
Scheme 3. Radiosynthesis of
K2CO3, DMF, 105 °C, 10 min.
[
18F]12. Reagents and conditions:
[
,
N
H
N
H
6 R = CH2CH2F
7 R = CH2C CH
8 R = CH2CH2F
9 R = CH2C CH
synthesized by a two-step labeling strategy using a [4 + 2] cycload-
dition between trans-cyclooctene and tetrazine.18,19 In vitro cell
studies and microPET tumor imaging using this tracer showed very
promising results.19,20 We now report a new radiolabeled PARP-1
inhibitor for measuring PARP-1 expression in vivo with PET. Based
on the benzimidazole carboxamide (NU1085)21 and its derivative
(AG014361),22 several analogs that could be easily labeled with
18F were synthesized, and their inhibition potency against PARP-1
was determined. 12 (IC50 = 6.3 1.3 nM) was labeled with 18F in
one step with high chemical and radiochemical purities. MicroPET
imaging demonstrated increased uptake of [18F]12 in MDA-MB-
436 tumors that was blocked by both 12 and olaparib/AZD2281.
O
NH2
N
N
N
F
(d)
N
O
N
H
10
Scheme 1. Synthesis of derivatives of NU1085 (2). Reagents and conditions: (a)
ROC6H4COCl (R = CH2CH2F for 6a and 6, R = CH2C„CH for 7a and 7) pyridine,
CH2Cl2; (b) CH3SO3H, MeOH; (c) NH3, MeOH; (d) 9, FCH2CH2N3, CuSO4, sodium
ascorbate, DMF.
H
H
H
N
N
O
N
O
O
NH
NH
N
N
R
O
(b)
NH
(a)
+
R
O
NH2
11
O
12
12a R = CH2CH2F
R = CH2CH2F
13a R = CH2C CH
14a R = CH2CH2Br
13 R = CH2C CH
14 R = CH2CH2Br
H
N
O
N
N
H
N
N
N
N
F
O
(c)
R
O
O
O
N
N
15
(d)
H
N
O
12 R = CH2CH2F
13 R = CH2C CH
14 R = CH2CH2Br
OMs
16
N
N
Scheme 2. Synthesis of derivatives of AG014361 (3). Reagents and conditions: (a) ROC6H4COCl (R = CH2CH2F for 12a and 12, R = CH2C„CH for 13a and 13, R = CH2CH2Br for
14a and 14), pyridine, CH2Cl2; (b) CH3SO3H, MeOH; (c) 13, FCH2CH2N3, CuSO4, sodium ascorbate, DMF; (d) 14, AgOMs, acetonitrile.

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D. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 1700–1707
Figure 2. Analytical HPLC of [18F]12, showing high chemical and radiochemical purities. (Top: UV, Bottom: radioactivity; specific activity = 11500 mCi/
lmol).
Figure 3. MicroPET images of MDA-MB-231 and MDA-MB-436 tumors in mice tumor at 60 min after [18F]12 injection. Top row: MDA-MB-231 tumor before and after
treatment with olaparib (ip 50 mg/kg 20 min pretreatment. Bottom row: MDA-MB-436 (right) and MDA-MB-231 (left) tumors before and after 12 (ip 1 mg/kg 20 min
pretreatment).
compound 6. After evaporation of the solvent, the mixture was re-
fluxed with methanesulfonic acid in methanol to give 6. Then the
methyl ester of 6 was converted to the amide compound 8 using
ammonia in methanol. Similarly, the alkyne analog 9 was synthe-
sized starting from 5 and 4-(prop-2-ynyloxy)benzoyl chloride. The
triazole compound 10 was prepared by the copper(I) catalyzed
click reaction of 2-fluoroethylazide and 9 using CuSO4Á5H2O and
sodium ascorbate in DMF.
2. Results
2.1. Chemistry
The syntheses of the new PARP-1 inhibitors are shown in
Schemes 1 and 2. Methyl 2,3-diaminobenzate (5) was reacted with
4-(2-fluoroethoxy)benzoyl chloride in pyridine and dichlorometh-
ane to afford a mixture of intermediate 6a and the benzimidazole

D. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 1700–1707
1703
Figure 4. Time-radioactivity curves of [18F]12 in MDA-MB-436 and MDA-MB-231 tumors in mice under baseline conditions and blocked with 12 (ip 1 mg/kg 20 min
pretreatment, top graph). The olaparib treated MDA-MB-231 tumor also demonstrated decreased [18F]12 uptake (bottom graph).
The tricycle analogs were synthesized from the diamine inter-
mediate 11. Compound 11 was reacted with 4-(2-fluoroeth-
oxy)benzoyl chloride in pyridine and dichloromethane to afford a
mixture of intermediate 12a and the benzimidazole 12. After evap-
oration of the solvent, the mixture was refluxed with methanesulf-
onic acid in methanol to give 12. Similarly, 13 and 14 were made
from the corresponding benzyl chlorides. Compound 15 was pre-
pared by the click reaction under the same condition as for 10
using 2-fluoroethylazide and 13. The mesylate precursor 16 for
the labeling of 12 with 18F was synthesized by reflux of 14 and sil-
ver methanesulfonate in acetonitrile.
in DMF at 105 °C (Scheme 3), affording [18F]12 in 40–50% yield (de-
cay corrected) after reversed phase HPLC purification and solid
phase extraction (Fig. 2). The total synthesis time was 90 min.
The specific activity of the final dose in 10% ethanol/saline was
5500–18,000 mCi/lmol.
2.3. MicroPET studies
MDA-MB-436 human breast cancer xenograft tumors in im-
mune-deficient mice were clearly visualized by PET using
[
18F]12. These tumors demonstrated increased uptake that was
Newly synthesized PARP-1 inhibitors were assessed for their
ability to inhibit active PARP-1 using the method described by Putt
and Hergenrother.23 The results are shown in Table 1. The tricycle
benzimidazole analogs had higher inhibition potency than their
respective benzimidazole analogs (e.g., 12 vs 8, 15 vs 10). In both
benzimidazole and tricycle benzimidazole analogs, the analogs
with fluoroethoxy substituent had three times higher inhibition
potencies than the respective analogs having a fluoroethyl triazole
group (e.g., 8 vs 10, 12 vs 15). Therefore, the most potent inhibitor,
12, was selected for 18F-labeling.
clearly distinguishable from background at 60 min post-tracer
injection (Fig. 3). The same mice treated with olaparib (50 mg/kg
ip) or 12 (1 mg/kg ip) 20 min prior to tracer injection decreased
[
18F]12 uptake in the tumors to the background tissue activity lev-
els (Fig. 3). The time–radioactivity curves of [18F]12 in the tumors
from 0 to 60 min of the microPET studies confirmed the visual
assessment of the microPET images (Fig. 4), demonstrating signif-
icantly decreased [18F]12 uptake as a result of treatment with
either olaparib or 12.
3. Discussion
2.2. Radiochemistry
Most PARP-1 inhibitors developed to date are NAD+ competitive
inhibitors.8,25 A large hydrophobic pocket adjacent to the nicotin-
amide binding site26 allows functionalization of these inhibitors
[
18F]12 was synthesized by the nucleophilic substitution of the
mesylate precursor 16 under conventional conditions (K222/K2CO3)

1704
D. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 1700–1707
to improve potency, solubility and to incorporate a radionuclide.
The benzimidazole carboxamide core is one of the most potent core
structures among reported PARP inhibitors.27 Therefore, we based
the design of the 18F-labeled PARP-1 inhibitors for PET imaging on
this core. Incorporation of the radionuclide is easily achieved either
through a fluoroethoxy group via nucleophilic substitution28 with
were reported as parts per million (ppm) downfield from tetra-
methylsilane (TMS). All coupling constants (J) are given in Hertz
(Hz). Splitting patterns are typically described as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. Elemental analysis (C, H, N) were
determined by Atlantic Microlab, Inc., Norcross, GA. High perfor-
mance liquid chromatography (HPLC) was performed with an
ultraviolet detector and a well-scintillation NaI (Tl) detector and
associated electronics for radioactivity detection. A Grace Altima
[
18F]fluoride or via Cu(I) catalyzed click reaction using 2-[18F]flu-
oroethyl azide.29,30 The corresponding 2-fluoroethoxy and 2-fluoro-
ethyl triazole analogs of NU1085 and AG014361 were respectively
synthesized according to Schemes 1 and 2 in only a few steps.
Although the propargyl and triazole groups slightly reduced the
inhibition potency of the analogs, the most potent of these were
12 (IC50 = 6.3 nM), an analog of AG014361 with a 2-fluoroethoxy
group, followed by 8 (IC50 = 10.8), an analog of NU1085. Therefore,
12 was chosen to be labeled for further evaluation in animals as a
PET tracer for imaging PARP-1 expression in vivo.
C18 250 Â 10 mm 10
l
semi-preparative column (A) and an Altima
analytical column (B) were used for prep-
C18 250 Â 4.6 mm 10
l
aration and analysis respectively. [18F]Fluoride was produced at
Washington University by the 18O(p,n)18F reaction through proton
irradiation of enriched (95%) [18O] water in the RDS111 cyclotron.
Radio-TLC was accomplished using a Bioscan AR-2000 imaging
scanner (Bioscan, Inc., Washington, DC). Published methods were
used for the synthesis of compound 527 and 11.22 All animal exper-
iments were conducted under Washington University Animal
Studies Committee IACUC-approved protocols in accordance with
the recommendations of the National Research Council’s ‘Guide
for the Care and Use of Laboratory Animals’.
The one-step labeling of [18F]12 under typical conditions can be
easily automated for clinical production of this tracer. The final
dose for injection had very high in chemical and radiochemical
purities and excellent specific activity. The injected mass of
[
18F]12 in the microPET studies is 0.0037–0.012
lg/dose (0.2 mCi/
dose), which is much lower than the blocking dose and therapeutic
doses. This amount of mass is unlikely to have a pharmacological
effect, and we anticipate that [18F]12 will be a useful PET tracer
for imaging PARP-1 expression.
5.2. Synthesis
5.2.1. Methyl 2-(4-(2-fluoroethoxy)phenyl)-1H-
benzo[d]imidazole-4-carboxylate (6)
MDA-MB-436 is a human breast cancer cell line with innately
high levels of PARP-1 activity19 and has been used in a mouse mod-
els to assess the efficacy of the 18F-labeled olaparib derivative for
imaging PARP-1 activity with microPET. [18F]12 progressively
accumulated in the tumor during the 1 h microPET acquisition,
and [18F]12 uptake was blocked in animals pretreated with either
olaparib or 12. Both olaparib and 12 are competitive PARP-1 inhib-
itors with high inhibition potencies (IC50 = 5 nM31 and 6.3 nM,
respectively). Thus, our data indicate that [18F]12 uptake in the
mouse model is due to PARP-1 expression, and that [18F]12 is a
promising PET tracer for in vivo imaging of PARP-1 expression
specifically.
A mixture of methyl 2,3-diaminobenzoate 5 (500 mg, 3 mmol)
and 4-(2-fluoroethoxy)benzoyl chloride (638 mg, 3.15 mmol) in
CH2Cl2 (10 mL) and pyridine (10 mL) was stirred overnight at
23 °C. After removal of the solvent under reduced pressure, the res-
idue was dissolved in methanol (50 mL), and followed by addition
of CH3SO3H (1 mL). After the mixture was refluxed for 3 h, metha-
nol was removed under reduced pressure, and the residue was dis-
solved in ethyl acetate (75 mL). The solution was washed with
saturated Na2CO3 (50 mL), water (50 mL) and saturated NaCl
(50 mL), and dried over Na2SO4. After evaporation of the solvent,
the crude product was purified by silica gel column chromatogra-
phy eluting with hexane–ethyl acetate (1:1) to afford 6 as white
solid (686 mg, 73%), mp 134.2–134.6 °C. 1H NMR (300 MHz, CDCl3)
d 10.58 (s, 1H), 7.98 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.7 Hz, 1H), 7.83
(d, J = 7.2 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 9.0 Hz, 2H),
4.75 (dt, J = 47.1 Hz, 4.2 Hz, 2H), 4.22 (dt, J = 27.6 Hz, 4.2 Hz, 2H),
3.96 (s, 3H). 13C NMR (75 MHz, CDCl3) d 167.0, 160.2, 152.3,
144.8, 135.0, 128.2, 124.4, 124.2, 122.3, 121.7, 114.9, 113.0, 81.6
(d, J = 169.7 Hz), 67.1 (d, J = 20.6 Hz), 52.0.
4. Conclusion
A highly potent PARP-1 inhibitor 12 (IC50 = 6.3 nM) has been
developed based on the tricycle benzimidazole core. [18F]12 was
synthesized using conventional labeling method with high purities
and specific activity. MicroPET studies of [18F]12 in MBA-MD-436
tumor-bearing mice demonstrated accumulation of the radioactiv-
ity in these tumors with known PARP-1 overexpression; the uptake
in the tumor was blocked by the highly potent PARP-1 inhibitor
olaparib and by 12, supporting the specificity of [18F]12 uptake
for PARP-1 activity. Therefore, [18F]12 is a promising PET tracer
for imaging PARP-1 expression in vivo that should be evaluated
for clinical utility.
5.2.2. Methyl 2-(4-(prop-2-ynyloxy)phenyl)-1H-
benzo[d]imidazole-4-carboxylate (7)
Compound 7 was prepared according to the same procedure for
compound 6, except using compound 5 (500 mg, 3 mmol) and 4-
(prop-2-ynyloxy)benzoyl chloride (613 mg, 3.15 mmol) as starting
materials. The crude product was purified by silica gel column
chromatography eluting with hexane–ethyl acetate (1:1) to afford
7 as white solid (724 mg, 79%), mp 176.0–176.8 °C. 1H NMR
5. Experimental
(300 MHz, CDCl3)
d 10.58, 8.03 (d, J = 9.0 Hz, 2H), 7.98 (d,
J = 8.1 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.10
(d, J = 9.0 Hz, 2H), 4.76 (d, J = 2.4 Hz, 2H), 4.00 (s, 3H), 2.57 (t,
J = 2.4 Hz, 1H). 13C NMR (75 MHz, CDCl3) d 167.1, 159.4, 152.4,
144.9, 135.1, 128.2, 124.6, 124.3, 122.8, 121.8, 115.4, 113.0, 77.9,
76.0, 55.8, 52.1.
5.1. General methods and materials
All chemicals were obtained from standard commercial sources
and used without further purification. All reactions were carried
out by standard air-free and moisture-free techniques under an in-
ert nitrogen atmosphere with dry solvents unless otherwise stated.
Flash column chromatography was conducted using Scientific
5.2.3. 2-(4-(2-Fluoroethoxy)phenyl)-1H-benzo[d]imidazole-4-
carboxamide (8)
A solution of 6 (315 mg, 1 mmol) in 7 N NH3 in methanol
(10 mL) was stirred for 3 days at 23 °C. After evaporation of the sol-
vent, the crude product was purified by silica gel column chroma-
tography eluting with hexane–ethyl acetate (1:2) to afford 8 as
Adsorbents, Inc. silica gel, 60A, ‘40 Micron Flash’ (32–63 lm). Melt-
ing points were determined using MEL-TEMP 3.0 apparatus and are
uncorrected. Routine 1H and 13C NMR spectra were recorded at
300 MHz on a Varian Mercury-VX spectrometer. All chemical shifts

D. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 1700–1707
1705
white solid (245 mg, 82%), mp 195.8–197.4 °C. 1H NMR (300 MHz,
DMSO-d6) d 9.44 (s, 1H), 8.23 (d, J = 9.0 Hz, 2H), 7.89 (d, J = 7.5 Hz,
1H), 7.80 (s, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.20
(d, J = 8.4 Hz, 2H), 4.81 (dt, J = 47.7 Hz, 3.6 Hz, 2H), 4.37 (dt,
J = 30.0 Hz, 3.9 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) d 166.4,
160.1, 152.0, 135.5, 128.6, 122.7, 122.0, 115.1, 82.1 (d,
J = 166.2 Hz), 67.3 (d, J = 19.4 Hz). Anal. Calcd for C16H14FN3O2.0.5-
H2O: C, 62.33; H, 4.90; N, 13.63. Found: C, 62.54; H, 4.87; N, 13.67.
starting materials. The crude product was purified by silica gel col-
umn chromatography eluting with ethyl acetate–methanol (10:1)
to afford 13 as white solid (268 mg, 84%), mp 258.0–259.1 °C. 1H
NMR (300 MHz, DMSO-d6) d 8.47 (s, 1H), 7.85 (m, 4H), 7.34 (t,
J = 7.8 Hz, 1H), 7.18 (d, J = 6.9 Hz, 2H), 4.93 (s, 2H), 4.46 (m, 2H),
3.64 (s, 1H), 3.54 (m, 2H). 13C NMR (75 MHz, DMSO-d6) d 167.4,
158.5, 153.6, 143.3, 132.5, 131.1, 125.1, 122.7, 122.4, 121.5,
117.7, 115.0, 79.0, 78.5, 55.6, 50.5. Anal. Calcd for C19H15N3O2: C,
71.91; H, 4.76; N, 13.24. Found: C, 71.71; H, 4.82; N, 12.98.
5.2.4. 2-(4-(Prop-2-ynyloxy)phenyl)-1H-benzo[d]imidazole-4-
carboxamide (9)
5.2.8. 2-(4-(2-Bromoethoxy)phenyl)-5,6-dihydro-imidazo[4,5,1-
jk][1,4]benzodiazepin-7(4H)-ones (14)
Compound 9 was prepared according to the same procedure for
compound 8, except using compound 7 (460 mg, 1.5 mmol) as
starting material. The crude product was purified by silica gel col-
umn chromatography eluting with hexane–ethyl acetate (1:2) to
afford 9 as white solid (378 mg, 86%), mp 183.4–183.9 °C. 1H
NMR (300 MHz, DMSO-d6) d 9.39 (s, 1H), 8.21 (d, J = 8.4 Hz, 2H),
7.87 (d, J = 7.8 Hz, 1H), 7.78 (s, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.32
(t, J = 7.8 Hz, 1H), 7.20 (d, J = 9.0 Hz, 2H), 4.92 (d, J = 1.8 Hz, 2H),
3.63 (s, 1H). 13C NMR (75 MHz, DMSO-d6) d 166.4, 159.0, 152.0,
128.5, 122.7, 122.3, 121.9, 115.4, 78.9, 78.6, 55.7. Anal. Calcd for
Compound 14 was prepared according to the same procedure
for compound 6, except using compound 11 (177 mg, 1 mmol)
and 4-(2-bromoethoxy)benzoyl chloride (277 mg, 1.05 mmol) as
starting materials. The crude product was purified by silica gel col-
umn chromatography eluting with ethyl acetate–methanol (10:1)
to afford 14 as white solid (255 mg, 66%), mp decomposed
280 °C. 1H NMR (400 MHz, DMSO-d6) d 8.44 (t, J = 5.6 Hz, 1H),
7.87 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.4 Hz,
2H), 7.34 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 4.44 (m, 4H),
3.86 (t, J = 5.2 Hz, 2H), 3.53 (m, 2H). 13C NMR (100 MHz, DMSO-
d6) d 167.8, 159.7, 154.1, 143.7, 132.9, 131.7, 125.5, 123.1, 122.6,
122.0, 118.1, 115.2, 68.4, 50.5, 40.8.
C
17H18N3O2Á0.5H2O: C, 67.99; H, 4.70; N, 13.99. Found: C, 67.97;
H, 4.72; N, 13.71.
5.2.5. 2-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-1H-benzo[d]imidazole-4-carboxamide (10)
A mixture of 9 (291 mg, 1.0 mmol), 1-azido-2-fluoroethane
(1.68 mmol), sodium ascorbate (990 mg, 5.0 mmol), and CuSO4-
Á5H2O (125 mg, 0.5 mmol) in DMF (10 mL) was stirred overnight
at 23 °C. The reaction mixture was diluted with ethyl acetate
(75 mL), and washed with water (2 Â 50 mL), and saturated NaCl
(50 mL), dried over Na2SO4. After evaporation of the solvent, the
crude product was purified by silica gel column chromatography
eluting with ethyl acetate to afford 10 as white solid (255 mg,
67%), mp 256.4–257.3 °C. 1H NMR (300 MHz, DMSO-d6) d 9.42 (s,
1H), 8.33 (s, 1H), 8.21 (d, J = 8.7 Hz, 2H), 7.87 (d, J = 7.5 Hz, 1H),
7.78 (s, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.27
(d, J = 8.7 Hz, 2H), 5.28 (s, 2H), 4.85 (dt, J = 46.8 Hz, 4.5 Hz, 2H),
4.76 (dt, J = 27.6 Hz, 4.2 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) d
166.3, 159.9, 152.0, 142.5, 141.6, 135.3, 128.6, 125.1, 122.7,
122.1, 121.9, 115.3, 114.7, 99.5, 81.9 (d, J = 167.3 Hz), 61.3, 50.1
(d, J = 20.5 Hz). Anal. Calcd for C19H17FN6O2: C, 59.99; H, 4.50; N,
22.09. Found: C, 60.10; H, 4.67; N, 21.49.
5.2.9. 5,6-Dihydro-2-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-imidazo[4,5,1-jk][1,4]benzodiazepin-
7(4H)-ones (15)
Compound 15 was prepared according to the same procedure for
compound 10, except using compound 13 (159 mg, 0.5 mmol) as
starting material. The crude product was purified by silica gel col-
umn chromatography eluting with ethyl acetate–methanol (10:1)
to afford 15 as white solid (147 mg, 72%), mp 226.5–227.6 °C. 1H
NMR (300 MHz, DMSO-d6) d 8.46 (t, J = 5.7 Hz, 1H), 8.33 (s, 1H),
7.89–7.81 (m, 4H), 7.34 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H),
5.28 (s, 2H), 4.85 (dt, J = 47.1 Hz, 4.2 Hz, 2H), 4.76 (dt, J = 27.6 Hz,
4.2 Hz, 2H), 4.45 (m, 2H), 3.54 (m, 2H). 13C NMR (75 MHz, DMSO-
d6) d 167.4, 159.4, 143.3, 142.6, 131.2, 125.1, 125.0, 122.7, 122.0,
121.5, 117.7, 114.8, 81.9 (d, J = 167.3 Hz), 61.2, 50.5, 50.1 (d,
J = 20.5 Hz), 40.4. Anal. Calcd for C21H19FN6O2.1.5H2O: C, 58.19; H,
5.12; N, 19.39. Found: C, 57.89; H, 4.54; N, 18.92.
5.2.10. 5,6-Dihydro-2-(4-(2-
(methylsulfonyloxy)ethoxy)phenyl)-imidazo[4,5,1-
5.2.6. 5,6-Dihydro-2-(4-(2-fluoroethoxy)phenyl)-imidazo[4,5,1-
jk][1,4]benzodiazepin-7(4H)-ones (12)
jk][1,4]benzodiazepin-7(4H)-ones (16)
A mixture of 14 (193 mg, 0.5 mmol) and AgOMs (508 mg,
2.5 mmol) was refluxed for 8 h. After evaporation of the solvent,
the crude product was purified by silica gel column chromatogra-
phy eluting with ethyl acetate–methanol (10:1) to afford 16 as
white solid (129 mg, 64%), mp 253.2–254.1 °C. 1H NMR
(400 MHz, CD3OD) d 7.97 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz,
1H), 7.76 (d, J = 8.8 Hz, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.17 (d,
J = 8.8 Hz, 2H), 4.59 (t, J = 4.0 Hz, 2H), 4.49 (t, J = 4.0 Hz, 2H), 4.35
(t, J = 4.0 Hz, 2H), 3.65 (m, 2H), 3.12 (s, 3H). 13C NMR (100 MHz,
CD3OD) d 160.2, 148.1, 142.7, 135.7, 132.2, 131.1, 131.0, 125.8,
122.6, 122.1, 121.4, 116.9, 114.6, 68.3, 66.0, 50.5, 40.6, 36.0.
Compound 12 was prepared according to the same procedure
for compound 6, except using compound 11 (177 mg, 1 mmol)
and 4-(2-fluoroethoxy)benzoyl chloride (213 mg, 1.05 mmol) as
starting materials. The crude product was purified by silica gel col-
umn chromatography eluting with ethyl acetate–methanol (10:1)
to afford 12 as white solid (247 mg, 76%), mp 236.0–237.5 °C. 1H
NMR (300 MHz, DMSO-d6) d 8.44 (t, J = 5.1 Hz, 1H), 7.89–7.80 (m,
4H), 7.34 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 8.7 Hz, 2H), 4.79 (dt,
J = 48.9 Hz, 3.6 Hz, 2H), 4.44 (m, 2H), 4.35 (dt, J = 31.2 Hz, 3.9 Hz,
2H), 3.53 (m, 2H). 13C NMR (75 MHz, DMSO-d6) d 167.8, 159.9,
154.1, 143.7, 132.9, 131.7, 125.5, 123.1, 122.5, 121.9, 118.1,
115.1, 82.5 (d, J = 165.0 Hz), 67.7 (d, J = 19.3 Hz), 50.9, 40.8. Anal.
Calcd for C18H16FN3O2: C, 66.45; H, 4.96; N, 12.92. Found: C,
66.43; H, 5.03; N, 12.92.
5.3. PARP-1. enzymatic assay
This assay is based on the chemical quantification of NAD+, that
is, the amount of NAD+ consumed when the active PARP-1 C-termi-
nal catalytic domain sequentially transfers ADP-ribose subunits
from nicotinamide adenine dinucleotide (NAD+) to protein
acceptors.7
5.2.7. 5,6-Dihydro-2-(4-(prop-2-ynyloxy)phenyl)-imidazo[4,5,1-
jk][1,4]benzodiazepin-7(4H)-ones (13)
Compound 13 was prepared according to the same procedure
for compound 6, except using compound 11 (177 mg, 1 mmol)
and 4-(prop-2-ynyloxy)benzoyl chloride (204 mg, 1.05 mmol) as
High-specific-activity PARP-1 and activated DNA were pur-
chased from Trevigen (Gaithersburg, MD). All other reagents re-

1706
D. Zhou et al. / Bioorg. Med. Chem. 22 (2014) 1700–1707
quired for this assay including NAD+ were purchased from Sigma–
Aldrich (St. Louis, MO). Known PARP-1 inhibitor PJ-34, used as a
control in these experiments, was synthesized in-house. To test
the compounds for PARP-1 inhibition, a solution of 250 nM NAD+
was first made in 50 mM Tris–HCl, 2 mM MgCl2, at pH 8.0 (PARP
for MEM and 5% FBS. Cells in exponential growth were trypsinized
and harvested for tumor implantation. After counting, cells were
re-suspended in ice-cold 1:1 Matrigel and PBS to give the desired
concentration and held on ice.
Mature female athymic nu/nu mice from Charles River Labora-
tories are allowed to acclimate in an AALAC accredited housing
facility for at least 1 week prior to tumor implantation for these se-
rial imaging studies. Female nu/nu mice were implanted in the
mammary fat pads (near the axillary lymph nodes) with 1 x 107
MDA-MB-436 breast cancer cells or either 1 Â 107 or 5 Â 106
assay buffer) and 20
fluorescence plate. A solution of 50
made in PARP assay buffer and 10 L was added to each well. Stock
solutions of test compounds were first prepared in DMSO, diluted
to varying concentrations in PARP assay buffer, and 10 L was
added to each well. To initiate the reaction, 10 L of 10 g/mL
PARP-1 enzyme in PARP assay buffer was added to each well.
The total volume was 50 L, bringing the final concentrations to
l
L transferred to each well of a 96-well black
lg/mL of activated DNA was
l
l
l
l
MDA-MB-231 breast cancer cells in ꢀ100
lL of 1:1 Matrigel:PBS.
Imaging studies were conducted 18–19 days post implantation.
Tumor-bearing mice were placed in an induction chamber con-
taining ꢀ2% isoflurane/oxygen and then secured to a custom dou-
ble bed for placement of tail vein catheters; anesthesia was
maintained via nose-cone at ꢀ1% isoflurane/oxygen for the dy-
namic imaging procedure. The mice were injected with 150–
l
2 lg/mL PARP-1 enzyme, 10 lg/mL activated DNA, and 100 nM
NAD+ per well. The plate was then incubated at room temperature
for 20 min. The amount of NAD+ present was then determined by
first adding 20
none (in ethanol) to each well. The plate was allowed to incubate
at 4 °C for 10 min. Then 90 L of 88% formic acid was added, result-
lL 2 M KOH, followed by 20 lL of 20% acetophe-
200 l
Ci of [18F]12 and scanned for 0–60 min using Focus 220 and
l
Inveon PET/CT scanners. The standard uptake values (SUVs) were
generated from manually drawn regions of interests for tumors
and surrounding ‘background’ tissue. Visualization of specific up-
take was determined by comparison of baseline scans with images
acquired by tracer injection 20 min after pre-treatment with the
blocking agents olaparib (50 mg/kg, ip) or 12 (1 mg/kg, ip).
ing in a final concentration of 222 mM KOH, 2.2% acetophenone,
44% formic acid, and varying concentrations of NAD+. The plate
was incubated at 100 °C for 5 min., allowed to cool, and then read
on a Perkin Elmer Victor microplate fluorometer (Waltham, MA)
using 360 nm excitation and 450 nm emission filters. Dose–re-
sponse curves were generated using GraphPad Prism version 5.04
for Windows (San Diego, CA) where control wells containing
NAD+ only were set at 0% PARP activity and control wells contain-
ing PARP-1 only were set at 100% PARP activity. IC50 values were
calculated from the dose–response curves generated from at least
Acknowledgments
NIH P01 HL13851 (PI: Gropler, Welch) supported this study.
The Doris Duke Foundation Clinical Investigator Award (PI: Chen)
and NIH R01 HL116389 (PI: Chen) supported DLC’s effort. We
thank Robert Dennett and Brian Wingbermuehle for the produc-
tion of [18F]fluoride, Justin Rothfuss for conducting the binding
affinity assays, and the staff of the Washington University Small
Animal Imaging Facility for their technical assistance.
three independent experiments and reported in Table
mean standard deviation (SD).
1 as
5.4. Radiosynthesis of [18F]12
[
18F]fluoride (up to 50 mCi in 100–500
transferred to a BD vacutainer (13 Â 75 mm, 5 mL, glass, no addi-
tives) containing K222 (2.2 mg, 5.8 mol) and K2CO3 (0.3 mg,
2.2 mol), then the mixture was dried by azeotropic distillation
l
L [18O]water) was
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