Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 213
Ta ble 3. Summary of Potentiation of Temozolomide (TM) and
Topotecan (TP) in LoVo Cell Line by 0.40 µM PARP-1 Inhibitor
Refer en ces
(1) From DNA Damage and Stress Signaling to Cell Death: Poly
ADP-ribosylation Reactions; de Murcia, G., Shall, S., Eds; Oxford
University Press: Oxford, 2000. Cell Death: The Role of PARP.;
Szabo, C., Ed.; CRC Press: Boca Raton, Fl, 2000 and references
therein.
(2) Griffin, R. J .; Curtin, N. J .; Newell, D. R.; Golding, B. T.; Durkacz
B. W.; Calvert, A. H. The role of inhibitors of poly(ADP-ribose)
polymerase as resistance-modifying agents in cancer therapy.
Biochimie 1995, 77, 408-422.
LoVo
compound
PF50 (TM)a
PF50 (TP)a
GI50 (µM)b
14
21
22
2.1
2.0
5.4
1.5
1.5
1.7
7.9
>50
11.2
a
b
See Table 1 and ref 7. GI50 of PARP-1 inhibitor alone against
(3) Masutani, M.; Nozaki, T.; Nishiyama, E.; Shimokawa, T.; Tachi,
Y.; Suzuki, H.; Nakagama, H.; Wakabayashi, K.; Sigimura, M.
Mol. Cell. Biochem. 1999, 193, 149-152. Menissier-de Murcia,
J .; Niedergang, C.; Trucco, C.; Ricoul, M.; Ditrillaux, B.; Mark,
M.; Olivier, F. J .; Masson, M.; Dierich, A.; LeMeur, M.; Walz-
tinger, C.; Chambon, P.; de Murcia, G. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 7303-7307. Wang, Z. Q.; Auer, B.; Stingl, L.;
Berghammer, H.; Haidacher, D.; Schweiger, M.; Wagner, E. F.
Genes Dev. 1995, 9, 509-520.
(4) Banasik, M.; Ueda, K. Inhibitors and activators of ADP-
ribosylation reactions. Mol. Cell. Biochem. 1994, 138, 185-197.
(5) Suto, M. J .; Turner, W. R.; Arundel-Suto, C. M.; Werbel, L. M.;
Sebolt-Leopold, J . S. Dihydroisoquinolinones: the design and
synthesis of a new series of potent inhibitors of poly(ADP-ribose)
polymerase. Anti-Cancer Drug Des. 1991, 7, 107-117.
(6) Delaney, C. A.; Wang, L. Z.; Kyle, S.; Srinivasan, S.; White, A.
W.; Calvert, A. H.; Curtin, N. J .; Durkacz, B. W.; Hostomsky,
Z.; Maegley, K.; Golding, B. T.; Griffin, R. G.; Newell, D. R.
Potentiation of temozolomide and topotecan growth inhibition
and cytotoxicity by novel poly(adenosine diphosphoribose) poly-
merase inhibitors in a panel of human tumor cell lines. Clin.
Cancer Res. 2000, 6, 2860-2867. Bowman, K. J .; Newell, D. R.;
Calvert, A. H.; Curtin, N. J . Differential effects of the poly(ADP-
ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase
I and II inhibitor cytotoxicity. Br. J Cancer 2001, 84, 106-112.
(7) White, A. W.; Almassy, R.; Calvert, A. H.; Curtin, N. J .; Griffin,
R. J .; Hostomsky, Z.; Maegley, K.; Newell, D. R.; Srinivasan, S.;
Golding, B. T. Resistance-modifying agents. 9. Synthesis and
biological properties of benzimidazole inhibitors of the DNA
repair enzyme poly(ADP-ribose) polymerase. J . Med. Chem.
2000, 43, 4084-4097.
LoVo cells.
in this study exhibited a broad array of cellular activity,
7,14
as measured by the potentiation factor PF50
(Table
1). Compounds 14 and 22 showed the largest amplifica-
tion of the cytotoxic effects of temozolomide (TM) and
topotecan (TP) against A549 cells (human lung carci-
noma). In contrast, the regioisomers of 14 and 22, 15
and 21, respectively, contain similar aryl groups and
have comparable Ki’s yet exhibit lower cellular efficacy.
To confirm inhibition of PARP-1 in intact cells, we
measured the NAD+ 15 and polymer levels16 within A549
cells. The cultures were initially treated with vehicle,
as control, to determine background levels. Subse-
quently, a set of cells were treated with the monofunc-
tional DNA alkylating agent N-methyl-N′-nitro-N-
nitrosoguanidine (MNNG) at subtoxic concentrations.
This gave control levels for NAD+ depletion and ADP-
ribose polymer formation resulting from DNA damage.
Finally, cells were treated with MNNG and 22 at
various concentrations. Table 2 shows the dose-depend-
ent ability of the inhibitor to suppress depletion of NAD+
and to inhibit polymer synthesis caused by chromosomal
damage. The compound 22 imparts a significant effect
even at 30 nM. Concurrent measurement of PF50 with
TP shows an analogous trend.
PARP inhibitors 14, 21, and 22 were also tested
against the LoVo cell line (colon cancer). All three
showed good levels of activity, with 22 again being the
most active (Table 3). These levels of potentiation were
obtained at 0.4 µM PARP inhibitor, which is well below
the cytotoxic level of the PARP-1 inhibitors alone (see
GI50’s in Table 3). By comparison, NU1085 (4) required
a concentration of 10 µM to achieve similar levels of
efficacy.7 This represents a ∼25-fold increase in chemo-
potentiation efficacy when compared to NU1085.
In summary, we have demonstrated a new and potent
class of PARP-1 inhibitors whose mode of action can be
directly linked to in vitro enzyme inhibition. Additional
preclinical studies will be reported in due course.
(8) Maryanoff, B. E.; Ho, W.; McComsey, D. F.; Reitz, A. B.; Grous,
P. P.; Nortey, S. O.; Shank, R. P.; Dubinsky, B.; Taylor, R. J .,
J r.; Gardocki, J . F. Potential Anxiolytic Agents. Pyrido[1,2-a]-
benzimidazoles: A New Structural Class of Ligands for the
Benzodiazepine Binding Site on GABA-A Receptors. J . Med.
Chem. 1995, 38, 16-20.
(9) Kamenka, J . M.; Alam, M. N. Synthesis in series of 6-oxoimidazo-
[4,5,1-ij]quinoline. J . Heterocycl. Chem. 1973, 10, 459-462.
(10) Kukla, M. J .; Breslin, H. J .; Pauwels, R.; Fedde, C. L.; Miranda,
M.; Scott, M. K.; Sherrill, Ronald G.; Raeymaekers, A.; Van
Gelder, J .; Andries, L.; J anssen, M. A. C.; De Clerq, R.; J anssen,
P. A. J . Synthesis and anti-HIV-1 activity of 4,5,6,7-tetrahydro-
5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one (TIBO)
derivatives. J . Med. Chem. 1991, 34, 746-751.
(11) We were unable to achieve cyclization with aliphatic acid
chlorides utilizing method A.
(12) Higgins, J .; Marvel, C. S. Benzimidazole polymers from aldeh-
dyes and tetraamines. J . Polym. Sci., Part A-1 1970, 8, 171-
177. Austen, S. C.; Kane, J . M. A short synthesis of the PARP
inhibitor 2-[4-(trifluoromethyl)phenyl]benzimidazole-4-carboxa-
mide (NU1077). J . Heterocycl. Chem. 2001, 38, 979-980.
(13) Marx, T.; Breitmaier, E. Chiral porphyrins with menthyl
residues attached to carbon. Liebigs Ann. Chem. 1992, 3, 183-
185.
(14) A PF50 value of 1.0 means there was no enhancement of the
cytotoxicity.
(15) J acobson, E. L.; J acobson, M. K. Tissue NAD as a biochemical
measure of niacin status in humans. Methods Enzymol. 1997,
280, 221-230.
Ack n ow led gm en t. We thank Suzanne Kyle and
Lan-Zhen Wang for their technical assistance in run-
ning potentiation assays.
Su p p or tin g In for m a tion Ava ila ble: Description of the
inhibition, potentiation, NAD+ depletion, and polymer forma-
tion assays, experiment detail and characterization for com-
pounds 6-25, and X-ray data for compound 22. This material
s.org.
(16) Aboul-Ela, N.; J acobson, E. L.; J acobson, M. K. Labeling methods
for the study of poly- and mono(ADP-ribose) metabolism in
cultured cells. Anal. Biochem. 1988, 174, 239-250.
J M0255769