Poly(ADP-ribose) Polymerase in Neurodegeneration: Radiosynthesis and Radioligand Binding in ARC-SWE tg Mice

Article abstract of DOI:10.1021/acschemneuro.8b00053

We report the synthesis, radiosynthesis, and characterization of a radioligand for poly(ADP-ribose) polymerase (PARP). PARP is of central importance in cell homeostasis, neuroplasticity, and neurodegeneration in the brain. A radiolabeled PARP inhibitor was developed and used for autoradiographic quantification of PARP protein concentration in wild-type and transgenic rodent brains ex vivo in high resolution. The binding of [³H]rucaparib was found to be confined to PARP-expressing domains, for example, cerebellar cortex or hippocampal regions in both models. Saturation binding experiments confirmed selective and reversible binding to a single site (K_d = 1.1 ± 0.2 nM).

Full text of DOI:10.1021/acschemneuro.8b00053

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Poly (ADP-ribose) polymerase in neurodegeneration:
Radiosynthesis and radioligand binding in ARC-SWE tg mice.
Santosh Reddy Alluri, and Patrick J Riss
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00053 • Publication Date (Web): 15 Mar 2018
Downloaded from http://pubs.acs.org on March 16, 2018
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Poly (ADP-ribose) polymerase in neurodegeneration: Radio-
synthesis and radioligand binding in ARC-SWE tg mice
Santosh R. Alluri,† Patrick J. Riss†‡
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†Realomics SFI, Kjemisk Institute, Universitetet i Oslo, Sem Sæalands Vei 26, Kjemibygningen, 0371, Oslo, Norway
‡Klinik for Kirurgi og Nevrofag, Oslo Universitets Sykehus HF−Rikshospitalet, Postboks 4950 Nydalen, 0424 Oslo, Norway
KEYWORDS: PARP, Rucaparib, Autoradiography
ABSTRACT: We report the synthesis, radiosynthesis and characterization of a radioligand for poly (ADPꢀribose) polymerase
(PARP). PARP is of central importance in cell homeostasis, neuroplasticity and neurodegeneration in the brain. A radiolabelled
PARP inhibitor was developed and used for autoradiographic quantification of PARP protein concentration in wildtype and transꢀ
genic rodent brains ex vivo in high resolution. The binding of [3H]rucaparib was found to be confined to PARPꢀexpressing domains
e.g. cerebellar cortex or hippocampal regions in both models. Saturation binding experiments confirmed selective and reversible
binding to a single site (Kd = 1.1±0.2 nM).
have been described. We decided to investigate a high affinity
PARPꢀ1 inhibitor as a novel radioligand to map PARP in brain
INTRODUCTION
The enzyme poly (ADPꢀribose) polymeraseꢀ1 (PARPꢀ1) is
in relation to neurodegenration and brain damage. Herein, we
one of the 17 members of the PARP family. It is a nuclear
describe the synthesis and radiosynthesis of [3H]rucaparib. A
enzyme that uses nicotinamide adenine dinucleotide (NAD+)
proof of principle study is conducted in a mouse model of
as its substrate to catalyze poly ADPꢀribosylation on acceptor
(amyloidogenic) dementia to test if lower PARP expression is
proteins.1 DNA damage activates PARPꢀ1, activated PARPꢀ1
detectable on the protein level. Concentration dependent bindꢀ
then adds ADPꢀribose to form a polymeric, energy rich scafꢀ
ing was determined to quantify PARP distribution using high
fold of polyꢀADPꢀribose) (PAR).1,2 PAR is a very critical
resolution autoradiography.
resource in cell homeostasis and repair. For example, base
excision repair (BER) and single strand break repair (SSBR)
mechanisms rely on PAR for energy. The roles of PARP in
disease are both ample and critical, mainly via disease mechaꢀ
RESULTS AND DISCUSSION
nisms involving PAR or PARP in their primary functions. The
most prominent include neuroplasticity, neuroprotecꢀ
tion/neurodegeneration and the main repair pathways in radiaꢀ
tion therapy.3,4,5 A PARP specific neurodegenerative mechaꢀ
nism, termed parthanatos, has been linked to PARP overactiꢀ
vation in response to DNA damage, which leads to cellular
depletion of NAD+, ATP, and, finally, nuclear translocation of
apoptosis inducing factor resulting in controlled cell death.6
Rucaparib (AG 014699) is a selective PARPꢀ1 inhibitor
used in clinical care. It has Ki value of 1.4 nM, which is the
highest value found in the literature.14 A crystal structure of
rucaparib bound to PARPꢀ1 active site has already been pubꢀ
lished which gave us confidence to use this compound to study
PARP in vivo. Very few syntheses of rucaparib 1 and its anaꢀ
logues e.g. 2 were reported. The key synthetic intermediate to
obtain rucaparib is the tricylic indolazepine 1f (Scheme 1).
We are interested in the role of PARP in long term memory
formation/synaptic plasticity.7,8 Alterations in PARylation
were shown to be implicated in changes transcripꢀ
tion/epigenetic patterns and thereby contribute to neurodegenꢀ
eration, for example in Alzheimer’s disease (AD).5,9ꢀ 10 PARyꢀ
lation on nuclear proteins is regarded as one of the chromatin
modifiers, the latter initiate new gene expression and protein
synthesis,8 and depletion of PARP signal in histochemical
studies was found in AD.11 Notably, PARP inhibition was
proven to be viable to neuroprotection in some animal modꢀ
els.12,13 Alterations in PARP activity were indicated in human
postmortem sections of Alzheimer’s brains.9,10
Scheme 1. Rucaparib and its synthetic intermediate
Few syntheses of 1f and its analog 2f has been reported in
good yield from a relatively expensive methyl 6ꢀfluoro indole
4ꢀcarboxylate (4 steps from 2ꢀmethyl 5ꢀfluoro benzoic acid)
over 3 steps.15,16,17 We devised a synthetic strategy that could
yield 1f over 4 steps from relatively cheap 6ꢀfluoro indole 1a
starting material via an intramolecular cyclization route
(Scheme 2). Rucaparib analog 2 was also synthesized using
this route along with 1.
Therefore we surmised that PARP protein concentration is
associated with longꢀterm synaptic changes including impaired
synaptic plasticity, tentatively even early in the disease. Howꢀ
ever, so far no studies on functional PARPꢀ1 protein and no
radioligands suitable for the measurements in high resolution
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Having 1d in hand, we proceeded further to prepare 1f. The
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Scheme 2. Synthetic route to 1f
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Schmidt rearrangement (HCl, NaN3) of 1d gave the desired 1e
in poor yields even using excess amount of reagents. Besides
the ketones 1d and 2d were also degraded to some extent in
acid media. Therefore 1d was first converted to its oxime
derivative and then subjected to Beckmann rearrangement to
obtain 1e (Supporting information, Scheme 2). In presence of
H2SO4, the oxime derivative of 1d gave a complex mixture of
products and 1e was difficult to isolate. It was assumed that
H2SO4 gave both the desired and undesired products due to
alkyl and aryl migration trans to nitrogen.20 Instead heating
the oxime derivative of 1d with SOCl2 gave the desired 1e as
the sole product in moderate 40% yield along with black tar.
The formation of 1e was confirmed by the coupling between
amide proton and adjacent –CH2 group protons (1HꢀCOSY
NMR). In the next step, the pivaloyl group was removed by
DBU and water to give 1f and 2f in very good yields.
Scheme 3: Synthesis of key intermediates 1f and 2f
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Table 1: Attempted syntheses to improve 1d yieldx
Reagents and conditions: i. (CH3CO)2O, CH3COOH, 50°C, 48 h,
1b 50%; ii. nꢀBuLi, ꢀ78°C, THF, 30 min, pivaloyl chloride, 1 h,
1c 80%, 2c 82%; iii.a) SOCl2, DCE, rt, 1 h, b)AlCl3, ClCH2COCl,
rt, 2 h, 1d 9%, 2d 71%; iv.a) NH2OH.HCl, CH3COONa.3H2O,
H2OꢀMeOHꢀTHF, 50°C, 2h, b) SOCl2, dioxane, 70°C, 6 h, 1e
40%, 2e 44%; v. DBU, water, THF, rt, 24 h, 1f 75%, 2f 80%
Entry indole 1c
Conditions
i.SOCl2
ii.AlCl3
i.SOCl2
ii.AlCl3
i.SOCl2
ii.AlCl3
t / h Product / %a
1
2
3
R, R1 = H
3
1j (70)
R= Piv
R1=H
R= Ts
R1=H
3
1d (9), 1j (57)
In the first step (Scheme 3), a propionic acid side chain was
installed at the carbon position 3 of 1a using acrylic acid and
acetic anhydride.18 Then the indole nitrogen of 1b, 2b was
protected with a pivaloyl group. The next key step in the synꢀ
thesis is a FriedelꢀCrafts acylation with intraꢀmolecular cyꢀ
clization. Teranishi and his coworkers19 had synthesized 2d
(Uhle’s ketone) from acyl chloride derivative of 2c using
AlCl3 and chloraacetyl chloride additive in 78% yield. The
electron withdrawing ability of the pivaloyl amide helps in
reducing nucleophilic activity of C2 and the tertꢀbutyl group
hinders C2 from electrophile attack which proved viable for
indoleꢀ3ꢀylpropionic acids. With 6ꢀfluoroindoleꢀ3ꢀylpropionic
acid, FriedelꢀCrafts acylation of 1c gave the desired 1d in 9%
yield which is quite low compared to that of 2d. Attempts
were made to improve the cyclization yield of 1d (Table 1) but
in most cases either the formation of side product 1j is noticed
or starting materials were degraded. The exception was found
in the entry 8 (40% yield, Supporting information, Scheme 3).
We presumed that the formation of 1j is due to the nucleoꢀ
philicity of the C2 position of the indole scaffold, here the
effect is more pronounced due to the negative inductive effect
of fluorine on the condensed benzo moiety. Changing the
protecting group from pivaloyl to tosyl or bulky TBDPS did
not improve the yield (Table 1, entries 3, 4). Neither using the
milder Lewis acid boron trifluoride nor the super strong triflic
acid (entries 5, 6) gave the desired product which limited our
means for optimization so that we decided to live with 9%
yield for 1d and proceed to make the final radioligand.
15
5
1j (24)
4
R=TBDPS i.SOCl2
ꢀ
R1=H
R=Piv
R1=H
R=ꢀPiv
R1=H
R=Ts
R1=Br
R=Ts
R1=Cl
ii.AlCl3
Triflic acid
0ꢀrt
5
10
16
5
ꢀ
6
i.SOCl2
ii.BF3(OEt)2
i.SOCl2
ii.AlCl3
i.SOCl2
ii.AlCl3
1j (18)
ꢀ
7b
8b
5
1r (40)
xReactions were performed with Indole (0.3 mmol), SOCl2 (1.2
mmol), AlCl3 (0.6 to 1.2 mmol), rt, DCE (5 mL), n≥2, aisolated
yield, TBDPS tertꢀbutyl diphenyl silyl, Ts tosyl; bSupporting
information
The next steps in the synthesis of 1 and 2 are bromination,
Suzuki coupling and reductive amination (Scheme 4). The
precursor for radiolabelling 1i was made from 1h via reductive
amination of aldehyde to primary amine and labelled with
radiomethylation reagent, [3H]methyl nosylate (Scheme 5).
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Scheme 4: Final synthesis of 1 and 2
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9
Reagent and conditions: i. Pyr.HBr3, DCMꢀTHF, 0°Cꢀrt, 2 h, 1g
69%, 2g 77%; ii. 4ꢀformaylphenylboronic acid, Pd(PPh3)4,
Na2CO3, dioxane, 90°C, 12 h, 1h 65%, 2h 70%; iii. a) 1M MeNH2
(MeOH), rt, 2 h, b) NaBH4, MeOHꢀTHF, 0°Cꢀrt, 1 66%, 2 71%
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Catalytic hydrogenation (Raney NiꢀH2, Pd/CꢀH2, Pd/Cꢀ
ammonium formate) of aldoxime derivative of 1h gave 1i in
low yields probably due to the debenzylation side reaction.
But using Zn dust and HCl, the aldoxime derivative of 1h gave
1i in very good yield.
Scheme 5: Synthesis of precursor 1i and [3H]1
O
O
O
S
CT3
H
H
N
N
H
O
N
O
O2N
O
i
NH
T
T
N
F
NH2
N
F
H
N
O
T
F
H
3
1
[ H]
H
1i
1h
Figure 1 Autoradiography images from saturation binding with
[3H]1 and competition with 1. (A) Wild type, [3H]1 total binding
(1 nM, control) (B) Comparison of [3H]1 binding in presence
(left), absence of 1µM of 1 (right) (C) Transgenic type, [3H]1
binding (1 nM, control) (D) Comparison of [3H]1 binding in
presence (left), absence of 1 µM of 1 (right) DG Dentate Gyrus
of hippocampus, CC Cerebellar cortex; n=3
Reagents, conditions; i. a) NH2OH.HCl, CH3COONa.3H2O, H2Oꢀ
MeOH, 50°C, 1 h, b) Zn, conc.HCl, EtOH, 0°Cꢀrt, 3 h, 1i 76%
A series of small scale (3 µmol) experiments (Supporting
information, Table 1) were performed to obtain the desired
monomethylated 1 and to suppress the formation of di/tri
methylated products under labeling conditions. The optimized
conditions were used to prepare [3H]1. [3H]methylation of 1i
was performed using [3H]methyl nosylate as methylatingꢀ
agent. The specific activity of [3H]1 is 82.8 Ci/mmol with
radiochemical purity 98.8% (Supporting information).
blotting and in situ hybridization methods have also provided
evidence on PARP mRNA expression in the dentate gyrus of
the hippocampus.24 Using Western blot analysis, Joanna et al,
reported PARP expression in hippocampus, cerebellum and
cerebral cortex regions.25 As it can be seen in Figure 1, the
addition of nonꢀradioactive 1 (1 µM) saturated the [3H]1 speꢀ
cific and selective binding in those regions, which highlights
the excellent nonꢀspecific binding of rucaparib. Concentration
dependent binding of [3H]1 was measured and the dissociation
constant (Kd) and Bmax were determined. The regional distribuꢀ
tion of [3H]1 is in good agreement with PARP distribution in
rodent brain.23ꢀ25,27ꢀ28
Autoradiography
At this point, we developed autoradiography conditions and
tested the PARP saturation by [3H]1 in two different types of
rodent brains, brains from wild type and transgenic mice carꢀ
rying the ArcꢀSwe double mutation were used.21,22 The brain
sections (0.02 mm thick) were thaw mounted onto a microꢀ
scopic slides and utilized for concentration dependent binding
of [3H]1 at increasing concentration of 1; see the supporting
information for experimental details. [3H]1 was found to bind
reversibly to a single site and allowed for quantification of
PARP in e.g. hippocampus and cerebellar cortex (Figure 1).
Figure 1 illustrates the PARP binding profile of [3H]1 in abꢀ
sence (A, C) and presence (B, D, left) of 1. The highest bindꢀ
ing densities of [3H]1 of all areas examined were found in the
hippocampal dentate gyrus and cerebellar cortex regions in
both the models (Figure 1 A,C). Areas such as cerebral cortex,
and thalamus were found to bind negligible amounts at all
radioligand concentrations proving low nonꢀspecific binding.
Nonꢀlinear regression analysis of the autoradiographic data
(Table 2) indicated that the binding was to single class of
binding sites in both key regions, the cerebellar cortex and the
dentate gyrus, implicated in PARPꢀrelated brain damage in
previous studies as explained above. Specific binding of [3H]1
was reproducibly saturable and nonspecific binding increased
with the concentrations of [3H]1 under the assay conditions
used. The Hill slopes of data derived from Prism program
were 0.75±0.1 suggesting that the ligand binds to one site with
no major cooperativity.26
When the binding of [3H]1 was examined between the two
different models under study, the transgenic models has shown
relatively lower PARP densities in the two regions. We preꢀ
sumed this reflects higher relative vulnerability of the transꢀ
The presence of PARP activity in hippocampal dentate gyꢀ
rus has been demonstrated previously using PAR in situ
(PARIS) or in situ end labeling techniques (ISEL).23 Northern
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genic animals to develop neurodegenerative phenotypes. Or
Conflicts of interest
1
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3
4
5
6
7
8
make them vulnerable to other insults to cell homeostasis
caused by the mutations. The PARP upꢀregulation also deꢀ
pends on the brain pathophysiology.27 Evidently, alterations in
PARP activity were indicated in human postmortem sections
of Alzheimer’s brains.9,10
None to be declared.
Notes
There is no competing financial interest
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Table 2 Saturation isotherm data
Type Region
Wild Cerebellar cortex 8.1±0.3
Dentate gyrus 7.2±0.5
Cerebellar cortex 7.4±0.4
Dentate gyrus 6.1±0.3
Bmax (pmol/mg) Kd (nM)
9
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fragmentation. Life Sci. 79, 2293–2302.
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(2015) Neuroprotection by the PARP inhibitor PJ34 modulates
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1.11±0.07
1.06±0.16
1.15±0.12
0.99±0.09
Tg
CONCLUSION
So far our understanding of the role of PARP in brain was
generated from histochemical studies on PARP activation or
RNAꢀexpression in rodent brains.23ꢀ25,27ꢀ28 The results deꢀ
scribed here with [3H]1 allow for direct assessment of PARPꢀ
protein binding by quantitative autoradiography to measure
[3H]1 binding to PARP protein throughout the brain. The
autoradiography provides quantitative measure of protein
density, which adds a new dimension to PARP related reꢀ
search. As a proofꢀofꢀprinciple we chose to measure PARP
saturation binding in brain regions associated with functional
PARPꢀ1 in wildtype and transgenic mice. The current observaꢀ
tions and the previous, related evidences may suggest PARP
association with neuronal damage,28,29 particularly in the hipꢀ
pocampal dentate gyrus. Dentate gyrus along with Olfactory
bulb and cerebellum are known to be the regions for neuroꢀ
genesis in adult brains.30ꢀ32 Proper pharmacological investigaꢀ
tions of functional PARP protein in brain may provide not
only a way to the neuroprotection but also a comprehensive
understanding to the relation between DNA damage/repair and
neurogenesis.
ASSOCIATED CONTENT
Supporting information
Experimental procedures and analytical data and supplemental
graphs. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Author contributions
S.R.A: Chemistry, autoradiography, data evaluation, manuscript
writing, supporting information compilation. P.J.R: Study design,
data evaluation, final manuscript compilation, funding
(17) Clark, R. D., Weinhardt, K. K., Berger, J., Fisher, L. E.,
Brown, C. M., MacKinnon, A. C., Kilpatrick, A. T., and Spedding, M.
(1990) 1,9ꢀAlkanoꢀbridged 2,3,4,5ꢀtetrahydroꢀ1Hꢀ3ꢀbenzazepines
with affinity for the alpha 2ꢀadrenoceptor and the 5ꢀHT1A receptor. J
Med Chem 33, 633–641.
Funds and acknowledgements
This study was funded by the Faculty of Mathematics and Natural
sciences, University of Oslo (PJR). Kjemisk institute, UiO is
thanked for the PhD fellowship (SRA).
Corresponding author Patrick J. Riss Tel: +47 22857673. E-mail:
patrick.riss@kjemi.uio.no
(18) Crosby, D. G. (1960) Joc 1969 25 569 1257, 569–570.
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(19) Teranishi, K., Hayashi, S., Nakatsuka, S. ichi, and Goto, T.
(1994) A facile biomimetic synthesis of uhle’s ketone by the
regioselective friedelꢀcrafts cyclization of indoleꢀ3ꢀylpropionyl
chloride. Tetrahedron Lett. 35, 8173–8176.
(20) Tatsuo Nagasaka, S. O. (1990) NIIꢀElectronic Library Service.
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