Identification of ring-fused pyrazolo pyridin-2-ones as novel poly(ADP-ribose)polymerase-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.091

A novel class of PARP-1 inhibitors was identified containing a non-aromatic heterocycle or carbocycle fused to a pyrazolo pyridin-2-one. Compounds displayed low nanomolar binding activity in the PARP-1 binding assay and submicromolar activity in a cell based chemosensitization assay.

Full text of DOI:10.1016/j.bmcl.2008.07.091

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5126–5129
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of ring-fused pyrazolo pyridin-2-ones as novel
poly(ADP-ribose)polymerase-1 inhibitors
b
b
b
b,
Wilna J. Moree a, , Phyllis Goldman , Anthony J. Demaggio , Erik Christenson , Dan Herendeen
,
*
John Eksterowicz a,à, Edward A. Kesicki b,§, David L. McElligott b, Graham Beaton a,–
a Deltagen Research Laboratories (Former CombiChem, Inc.), 4570 Executive Drive, Suite 400, San Diego, CA 92121, USA
b ICOS Corporation (acquired by Eli Lilly and Company), 22021 20th Avenue S.E., Bothell, WA 98021, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of PARP-1 inhibitors was identified containing a non-aromatic heterocycle or carbocycle
fused to a pyrazolo pyridin-2-one. Compounds displayed low nanomolar binding activity in the PARP-
1 binding assay and submicromolar activity in a cell based chemosensitization assay.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 27 May 2008
Revised 23 July 2008
Accepted 24 July 2008
Available online 27 July 2008
Keywords:
PARP-1 inhibitor
Cancer
DNA repair
Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant
nuclear enzyme that is activated by single or double DNA strand
breaks.1 Activated PARP-1 uses nicotinamide adenine dinucleotide
as a substrate to catalyze the addition of long branched chains of
poly ADP ribose onto itself and other target proteins involved in
DNA replication, transcription, and repair.2
studied as protective agents against tissue damage occurring from
ischemia/reperfusion injuries5 and various forms of inflammation.6
The vast majority of PARP-1 inhibitors described to date are
competitive with NAD+, are analogs of nicotinamide, and have a
carboxamide attached to an aromatic ring or contain a fused aro-
matic lactam (Fig. 1).7
The involvement of PARP-1 in the regulation of DNA integrity
has made it an attractive target for cancer therapy. PARP-1 has
been demonstrated to be important for repair of damage induced
by radiation as well as by different classes of chemotherapy agents,
including the topoisomerase I inhibitors and the mono-functional
DNA alkylating agents. Disruption of PARP-1 expression or func-
tion results in increased sensitivity of tumor cells to these cancer
agents.3 Indeed, PARP-1 inhibitors have been in development as
sensitizers in radiation and chemotherapy treatment of cancer
for many years.4 In addition, PARP-1 inhibitors are currently being
In this letter, we describe the discovery, synthesis, and SAR of a
novel series of tricyclic PARP-1 inhibitors that contains a non-aro-
matic heterocycle or carbocycle fused to a pyrazolo pyridin-2-one.8
To initiate this program, a large collection of commercial sam-
ples from different vendors was screened in a PARP-1 binding as-
say. From this screening effort, compound 5 was identified as a
potent PARP-1 inhibitor with an IC50 of 93 nM (Fig. 2). However,
after resynthesis of this compound and some close analogs, no sig-
nificant binding affinity for PARP-1 was observed. NMR analysis of
the commercial sample 59 showed strong NOE enhancements be-
tween methyl protons and methylene protons suggesting that
the correct structure was in fact compound 6, containing a pyri-
din-2-one (Fig. 2). This pharmacophore with a s-trans conforma-
tion of an amide functionality is known to be a favorable binding
conformation for PARP-1 inhibitors as described in the litera-
ture.10,11 Close analogs of structure 6 were synthesized, varying
both the substituents off the pyrazole unit and the non-aromatic
heterocycle fused to the pyridin-2-one.
* Corresponding author at present address: Neurocrine Biosciences, Inc., 12780 El
Camino Real, San Diego, CA 92130, USA. Tel.: +1 858 617 7506; fax: +1 858 617
7925.
E-mail addresses: wmoree@neurocrine.com (W.J. Moree), echristenson@comcast.
net (E. Christenson), dmcelligott@borealisbiotech.com (D.L. McElligott).
Present address: Koronis Pharmaceuticals, 12277 134th Court NE, Redmond, WA
98052, USA.
à
Two synthetic routes were employed for the synthesis of ana-
logs around compounds 5 and 6:
Method A: Cyclic b-ketoesters (7) were reacted with 5-amino-
pyrazoles (8) in PPA at ꢀ130 °C to generate pyridin-4-ones pre-
dominantly12 (9) (Scheme 1). If reaction times surpassed 15 min
Present address: Amgen, 1120 Veterans Boulevard, South San Francisco, CA
94080, USA.
§
Present address: Afya World Medicines, 1124 Columbia Street, #600, Seattle, WA
98104, USA.
–
Present address: Neurocrine Biosciences, Inc., 12780 El Camino Real, San Diego,
CA 92130, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.091

W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5126–5129
5127
O
N
O
O
O
NH
NH2
NH
NH
HN
HN
HN
N
O
N
1
2
3
4
OMe
Figure 1. PARP-1 inhibitors 1, 2, 3, and 4 with PARP-1 IC50 values of 160, 60, 20, and 5 nM, respectively.
for the tetrahydrothiophene subseries, significant aromatization to
the thiophene pyridine-4-one occurred. When cyclic b-ketoesters
(7) were reacted with 5-aminopyrazoles (8) under basic condi-
tions13 amide intermediates (10) were formed which were purified
by HPLC chromatography. Subsequent cyclization under acidic
conditions or at high temperature in vacuo gave the pyrazolo pyri-
din-2-ones (11). In early reports, it is described that the enamine
condensation product from 5-amino-1,3-dimethylpyrazole and
ethyl acetoacetate does not cyclize in acetic acid to yield the ex-
pected 4-pyridinone but rather the 2-pyridinone due to intercon-
version of the enamine intermediate to the amide condensation
product (10).14 This possibly led to the disparity in assignment of
the commercial HTS lead (5).
was observed with bulky substituents at R1 such as t-butyl or phe-
nyl in the presence of methyl at R2 (19, 20, 24, 25, 30). Phenyl sub-
stituents at both R1 and R2 (26, 31) caused a complete loss of
inhibitory activity.
Preliminary docking experiments using the co-crystal structure
of chicken PARP-1 (1PAX) and the Parke-Davis/Pfizer inhibitor11a
(1) indicated that the pyrazolo pyridin-2-ones have a similar bind-
ing mode19 and provided a rationalization for the observed PARP-1
data. In Figure 3, a high scoring pose of compound 22 is shown,
making two hydrogen bonds between the amide bond and Gly-
863 and Ser 904. A fairly large binding pocket can accommodate
the phenyl group or a variety of other substituents at R2 in subser-
ies II. The volume tolerance in the direction of R1 in subseries II was
substantially less. Figure 3 displays a high scoring pose of inactive
compound 25. In this orientation the amide can only make one of
the two key donor/acceptor interactions because of the orientation
required to accommodate the R1 phenyl group (subseries II) in the
active site.
Comparison of dihydrothiophene (I), cyclopentene (II) and
cyclohexene (III) sub-series, indicates a preference for binding
affinity according to cyclohexene III > cyclopentene II > dihydrothi-
ophene I. These results are consistent with the predicted outcome
from modeling. Docking shows subseries III to be the most favored
as the additional methylene from the cyclohexyl provides addi-
tional favorable hydrophobic contact with PARP-1. In a comparison
of 6, 21, and 27, the hydrophobic surface area is 7% larger for 27
than for 6 and 21 and the ligand/protein contact in this area of
the binding site is predominantly aromatic/hydrophobic. A similar
Method B by Winters et al. 15: Cyclic ketones (12) were reacted
with 5-aminopyrazoles (8) to give dienamines (13), converted to
ureas and subjected to thermal cyclization to give pyrazolo pyri-
din-2-ones (14) (Scheme 2).16 When tetrahydrothiophen-3-one
was used in this sequence, the major product isolated upon con-
densation with 5-aminopyrazole (8) was a fully aromatized thio-
phene aminopyrazole (13). Therefore this method was only
employed with the carbocyclic ketones. All compounds were iso-
lated by either filtration from crude reaction mixtures or reverse
phase HPLC/MS purification using mass triggered fraction
collection.17
Compounds were evaluated in a PARP-1 binding assay,18 and
the results are summarized in Tables 1–4. Representatives of the
pyridin-4-one series (Table 1), including 5, the reported structure
of the high-throughput screening hit, did not display the inhibitory
activity for PARP-1 as one would have expected based on the re-
sults (IC50 = 93 nM) for the commercial sample. None of the other
compounds in this series, in which the pyrazolo substituents and
the non-aromatic heterocycle attached to the pyridin-4-one core
O
R2
N
X = S, CH2 , -CH2CH2-
were varied, showed PARP-1 inhibition with IC50 values <5
(data not shown).
lM
X
a
N
R1
NH2
In contrast, the pyridin-2-one series resulted in potent PARP-1
inhibitors (Tables 2–4). In the dihydrothiophene (I), cyclopentene
(II) and cyclohexene (III) subseries, the most active compounds
contained methyl substituents at both R1 and R2 positions (6, 21,
27). Replacement of the methyl at R2 by phenyl or 2-thienyl typi-
cally resulted in a small decrease (up to 5-fold) in inhibition (17,
18, 22, 23, 28, 29). A more dramatic decrease in binding affinity
N
H
O
O
R1
OR
N
N
9
X
+
R2
d
O
7
8
R1
X = S
NH
X
N
O
N
O
R2
10
NH
b or c
X = S
X
10
R1
N
N
R2
11
Scheme 1. Reagents and conditions: (a) PPA, 130 °C, 2 h (X = CH2 and CH2CH2),
15 min (X = S); (b) HOAc, reflux, 1 h; (c) ꢀ220 °C, vacuo, 15 min; (d) Pyr, o-Xylene,
110 °C, 2–6 h.
Figure 2. Reported structure (5) and correct structure (6) of high-throughput
screening hit.

5128
W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5126–5129
O
n=1,2
n=1,2
n=1,2
NH2
NH
b, c
a
8
+
R1
O
R1
N
N
N
N
R2
R2
14
12
13
Scheme 2. Reagents and conditions: (a) HOAc, rt, 45 h; (b) PhN@C@O, toluene, 3 h, rt then 15 minÀ3 h at 85 °C (c) ꢀ200 °C, 5–15 min.
Table 1
Table 3
PARP-1 inhibition for pyrazolo pyridin-4-ones
PARP-1 inhibition for cyclopentene subseries II
O
O
NH
N
II
N
X
R1
N
N
H
N
R2
Compound
X
PARP-1 Inhibition IC50, l
Ma
Compound
R1
R2
PARP-1 Inhibition IC50a, nM
5
15
16
–S–
–CH2–
–CH2CH2–
>10
7
>10
21
22
23
24
25
26
Me
Me
Me
t-Bu
Ph
Me
Ph
29.8
106.0
148.6
519.2
>10,000
>10,000
2-Thienyl
Me
a
Values for single experiments.
Me
Ph
Ph
increase in binding potency was observed in a bicyclic uracil series
when a cyclopentyl group was replaced by a cyclohexyl moiety.20
Potent compounds were subsequently tested for their ability to
augment cytotoxicity of the alkylating agent streptozotocin with
the human colon carcinoma HCT 116 cell line21 (Table 5). Com-
Single determinations when IC50 > 1000 nM.
a
Values of duplicate determinations are within twofold of each other.
pounds from the cyclohexene subseries III, the most potent in
the enzyme binding assay (27–29), were also most active in the
Table 2
Table 4
PARP-1 inhibition for dihydrothiophene subseries I
PARP-1 inhibition for cyclohexene subseries III
O
O
NH
NH
I
III
S
R1
R1
N
N
N
N
R2
R2
R1
R2
PARP-1 Inhibition IC50a, nM
Compound
R1
R2
PARP-1 Inhibition IC50a, nM
Compound
27
28
29
30
31
Me
Me
Me
Ph
Me
Ph
2-Thienyl
Me
Ph
2.2
7.3
6.9
9720
>10,000
6
Me
Me
Me
t-Bu
Ph
Me
Ph
2-Thienyl
Me
92.7
17
18
19
20
110.3
428.1
1841.5
>10,000
Ph
Me
Single determinations when IC50 > 1000 nM.
Single determinations when IC50 > 1000 nM.
a
a
Values of duplicate determinations are within twofold of each other.
Values of duplicate determinations are within twofold of each other.
Figure 3. Dock of 22 (left) and 25 (right) using the co-crystal structure of chicken PARP-1 (1PAX) and the Parke-Davis/Pfizer inhibitor (1).

W. J. Moree et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5126–5129
5129
Table 5
Menissier-de Murcia, J.; de Murcia, G. Mol. Cell. Biochem. 1999, 193, 53; (g)
Wang, Z.; Auer, B.; Stingl, L.; Berghammer, H.; Haidacher, D.; Schweiger, M.;
Wagner, E. F. Genes Dev. 1995, 9, 509; (h) Sebolt-Leopold, J. S.; Scavone, S. V. Int.
J. Radiat. Oncol. Biol. Phys. 1992, 22, 619.
Chemosensitization, cell toxicity, and therapeutic index for compounds 6 and 17, 21–
23, and 27–29
Compound
ECsens
,
l
Ma
LD40
>50
,
lM
TIc
4. (a) Borek, C.; Morgan, W. F.; Ong, A.; Cleaver, J. E. Proc. Natl. Acad. Sci. U.S.A.
1984, 81, 243; (b) Suto, M. J.; Suto, C. M. Drugs Future 1991, 16, 723; (c) Griffin,
R. J.; Curtin, N. J.; Newell, D. R.; Golding, B. T.; Durkacz, B. W.; Calvert, A. H.
Biochimie 1995, 77, 408; (d) Miknyoczk, S. J.; Jones-Bolin, S.; Pritchard, S.;
Hunter, K.; Zhao, H.; Wan, W.; Ator, M.; Bihovsky, R.; Hudkins, R.; Chatterjee, S.;
Klein-Szanto, A.; Dionne, C.; Ruggeri, B. Mol. Cancer Ther. 2003, 2, 371; (e)
Calabrese, C. R.; Almassy, R.; Barton, S., et al J. Natl. Cancer Inst. 2004, 96, 56; (f)
Madhusudan, S.; Middleton, M. R. Cancer Treat. Rev. 2005, 31, 603.
5. (a) Zhang, J. Emerging Drugs 1999, 4, 209; (b) Sharp, C.; Warren, A.; Oshima, T.;
Williams, L.; Li, J. H.; Alexander, J. S. Inflammation 2001, 25, 157.
6
17
21
22
23
27
28
29
28.6
15.4
19.3
26.8
3.1 (1.3)
1.9 (1.2)
>1.8
1.4
1.4
1.6
3.6
>6.6
19.7
>32.9
21.2
27.2
42.5
11.2
>12.5b
7.5b
0.38 (0.15)
0.38 (0.21)
>12.5b
6. (a) Virag, J.; Bai, P.; Bak, I. Med. Sci. Monit. 2004, 10, BR77; (b) Liaudet, L.; Pacher,
P.; Mabley, J. G. Am. J. Respir. Crit. Care Med. 2002, 165, 372; (c) Veres, B.; Radnai,
B., ; Gallyas, F., Jr.; Varbiro, G.; Berente, Z.; Osz, E.; Sumegi, B. J. Pharmacol. Exp.
Ther. 2004, 310, 247; (d) Chiarugi, A. Br. J. Pharmacol. 2002, 137, 761.
7. For reviews see (a) Li, J.-H.; Zhang, J. Idrugs 2001, 4, 804; (b) Cosi, C. Expert Opin.
Ther. Patents 2002, 12, 1047; (c) Southan, G. J.; Szabo, C. Curr. Med. Chem. 2003,
10, 321; (d) Peukert, S.; Schwahn, U. Exp. Opin. Ther. Pat. 2004, 14, 1531.
8. Preliminary data of this series were first disclosed in: Moree, W. J.; Goldman, P.;
Demaggio, A. J.; Christenson, E.; Herendeen, D.; Eksterowicz, J.; Kesicki, E. A.;
McElligott, D. L.; Beaton, G.; Poster presentation at the 28th Medicinal
Chemistry Symposium, San Diego, CA, USA, June 8–12, 2002.
a
Values for quadruplicate experiments. Single determinations if ECsens values
were >10 uM.
b
c
Values of duplicate determinations are within twofold of each other.
In vitro therapeutic index (LD40/ECsens).
chemosensitization assay and displayed the most favorable thera-
peutic index. Addition of an aromatic group at R2 in compounds 28
and 29 appeared to accentuate chemosensitizer potency relative to
the dimethyl analog 27. Since analogs 27-29 exhibited similar en-
zyme potency in vitro, this result implies that other factors are
contributing to the enhanced potency of the aryl-substituted ana-
logs in the cell-based assay. Possible factors would include solubil-
ity and cellular permeability, which is consistent with the observed
lack of activity for compound 21 in cyclopentene subseries II.
In conclusion, tricyclic pyrazolo pyridin-2-ones containing a
non-aromatic carbocyle or heterocycle fused to the pyridin-2-one
were identified as a novel series of potent low nanomolar inhibi-
tors of PARP-1. Structure activity was further developed in the pyr-
azolo moiety resulting in the identification of analogs with
submicromolar activity in a chemosensitization assay.
9. 1H NMR (d6-DMSO) d 2.34 (3H, s, CH3–C), 3.77 (3H, s, CH3N), 3.97 (2H, t,
J = 3.2 Hz, CH2), 4.51 (2H, t, J = 3.2 Hz, CH2), 12.17 (1H, bs, NH). NOE observed
between s at 2.34 ppm and t at 4.51 ppm.
10. Suto, M. J.; Turner, W. R.; Arundel-Suto, C. M.; Werbel, L. M.; Sebolt-Leopold, J.
S. Anti-Cancer Drug Des. 1991, 6, 107.
11. (a) Ruf, A.; Menissier-de Murcia, J.; de Murcia, G. M.; Schulz, G. E. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 7481; (b) Ruf, A.; de Murcia, G. M.; Schulz, G. E.
Biochemistry 1998, 37, 3893; (c) Costantino, G.; Macchiarulo, A.; Camaioni, E.;
Pellicciari, R. J. Med. Chem. 2001, 44, 3786.
12. Kappe, T.; Zadeh, R. K. Synthesis 1975, 4, 247.
13. Raban, M.; Martin, V. A.; Craine, L. J. Org. Chem. 1990, 55, 4311.
14. Ratajczyk, J. D.; Swett, L. R. J. Heterocycl. Chem. 1975, 12, 517.
15. (a) Winters, G.; Sala, A.; De Paoli, A.; Conti, M. Synthesis 1984, 12, 1050; (b)
Winters, G.; Sala, A.; De Paoli, A.; Ferri, V. Synthesis 1984, 12, 1052.
16. Synthesis of compound 28: 4-Cyclohex-1-enyl-2-methyl-5-phenyl-2H-
pyrazol-3-ylamine (13, n = 2, R1 = Me, R2 = Ph), prepared as described by
Winters et al.15a (50 mg, 0.20 mmol), was suspended in 800
treated with phenylisocyanate (26
l
l toluene and
l
l, 0.24 mmol).15b After stirring at rt for 3 h
The compounds described herein could be useful for further
studies on the role of PARP-1 inhibitors as adjunct agents in cancer
therapy.
and heating at 85 °C for 2.5 h, the mixture was concentrated in vacuo to give a
yellow oil. The crude product was heated at 200 °C for 5 min, cooled down to rt
and triturated with EtOAc (400 lL). The precipitate was filtered and washed
with EtOAc to yield 41 mg of 28 (72%). MS: m/z 280 [M+H]+, expected 280
[M+H]+. 1H NMR (d6-DMSO) d 1.46–1.56 (2H, m, CH2), 1.62–1.70 (2H, m, CH2),
2.36–2.47 (4H, m, CH2), 3.85 (3H, s, CH3N), 7.40–7.57 (5H, m, Ph), 8.63 (1H, bs,
NH).
Acknowledgments
17. Zeng, L.; Kassel, D. B. Anal. Chem. 1998, 70, 4380.
18. The ability of compounds to inhibit PARP-1 activity was tested in an assay
using PARP-1 enzyme isolated from HeLa cells. An eleven-point dilution series
The authors thank Dr. Mark J. Suto, Dr. Peter L. Myers, Dr. Kerry
W. Fowler and Dr. Michael Gallatin for support of this program, Dr.
Catherine Jolivet for NMR analysis, and Mr. Tao Wang for analytical
assistance.
ranging from 10 to 0.01
lM was prepared in singlet for each compound. Each
compound dilution was mixed with 100 ng of PARP-1 enzyme, 33 ng sheared
E. Coli Stain B Type VIII DNA (Sigma), 2.5 lM cold NAD (Sigma), and 2 lCi
[adenylate-32P]-NAD (NEN). The reaction components were incubated at room
temperature for 10 min, at which time the reactions were stopped with a
twofold volume of saturated ammonium sulfate. Reactions were filtered over
MAIP filter plates (Millipore), scintillation fluid was added, and the plates were
counted. IC50 were determined from the dilution curves.
References and notes
1. Pieper, A. A.; Verma, A.; Zhang, J.; Snyder, S. H. Trends Pharmacol. Sci. 1999, 20,
171.
19. Glide, version 4.0, Schrödinger, LLC New York, NY, 2005.
20. Steinhagen, H.; Gerisch, M.; Mittendorf, J.; Schlemmer, K-H.; Albrecht, B.
Bioorg. Med. Chem. Lett. 2002, 12, 3187.
21. The ability of PARP inhibitors to augment the cytotoxicity of the alkylating
agent streptozotocin (ICN Pharmaceuticals) was tested in the human colon
carcinoma HCT116 cell line (ATCC). PARP inhibitors and streptozotocin were
diluted in culture media and added to 96-well plates. HCT116 cells were
trypsinized and seeded into the plates at a final concentration of 1000 cells/
well. Plates were incubated in a 37 °C, 5% CO2 incubator for four days. Cells
were then labeled with [methyl-3H]-thymidine (NEN) at a concentration of
2. (a) Menissier-de Murcia, J.; Molinete, M.; Gradwohl, G.; Simonin, F.; de Murcia,
G. J. Mol. Biol. 1989, 210, 229; (b) Gradwohl, G.; Menissier-de Murcia, J.;
Molinete, M.; Simonin, F.; Koken, M.; Hoeijmakers, J. H. J.; de Murcia, G. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 2990; (c) Zahradka, P.; Ebisuzaki, K. Eur. J.
Biochem. 1984, 142, 503.
3. (a) Menissier-de Murcia, J.; Niedergang, C.; Trucco, C.; Ricoul, M.; Dutrillaux, B.;
Mark, M.; Oliver, F. J.; Masson, M.; Dierich, A.; LeMeur, M.; Walztinger, C.;
Chambon, P.; de Murcia, G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7303; (b)
Eliasson, M. J. L.; Sampei, K.; Mandir, A. S.; Hurn, P. D.; Traystman, R. J.; Bao, J.;
Pieper, A.; Wang, Z.-Q.; Dawson, T. M.; Snyder, S. H.; Dawson, V. L. Nature Med.
1997, 3, 1089; (c) Mabley, J. G.; Suarez-Pinzon, W. L.; Hasko, G.; Salzman, A. L.;
Rabinovitch, A.; Kun, E.; Szabo, C. Br. J. Pharmacol. 2001, 133, 909; (d) Masutani,
M.; Nozaki, T.; Nishiyama, E.; Shimokawa, T.; Tachi, Y.; Suzuki, H.; Nakagama,
H.; Wakabayashi, K.; Sugimura, T. Mol. Cell. Biochem. 1999, 193, 149; (e)
Masutani, M.; Nozaki, T.; Nakamoto, K.; Nakagama, H.; Suzuki, H.; Kusuoka, O.;
Tsutsumi, M.; Sugimura, T. Mutat. Res. 2000, 462, 159; (f) Trucco, C.; Rolli, V.;
Oliver, F. J.; Flatter, E.; Masson, M.; Dantzer, F.; Niedergang, C.; Dutrillaux, B.;
1 lCi/well. The plates were incubated for another 24 h at 37 °C, 5% CO2. The
plates were harvested and then counted using a Matrix 96 Direct Beta Counter
(Packard). To compare the potency of PARP inhibitors, the concentration of
PARP inhibitor was calculated that reduced by half the amount of
chemotherapy agent required for 90% inhibition of cell growth (ECsens). The
concentration of PARP inhibitor that is significantly toxic as a single agent
(LD40) was also determined.