Potentiation of cytotoxic drug activity in human tumour cell lines, by amine-substituted 2-arylbenzimidazole-4-carboxamide PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.03.017

The synthesis and biological evaluation of a new series of amine-substituted 2-arylbenzimidazole-4-carboxamide inhibitors of the DNA-repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is reported. The introduction of an amine substituent at the 2-aryl position is not detrimental to activity, with most inhibitors exhibiting K_i values for PARP-1 inhibition in the low nanomolar range. Two compounds in this series were found to potentiate the cytotoxicity of the DNA-methylating agent temozolomide by 4-5-fold in a human colorectal cancer cell line.

Full text of DOI:10.1016/j.bmcl.2004.03.017

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2433–2437
Potentiation of cytotoxic drug activity in human tumour cell lines,
by amine-substituted 2-arylbenzimidazole-4-carboxamide
PARP-1 inhibitors
Alex W. White,a Nicola J. Curtin,b Brian W. Eastman,c Bernard T. Golding,a
Zdenek Hostomsky,c Suzanne Kyle,b Jianke Li,c Karen A. Maegley,c Donald J. Skalitzky,c
Stephen E. Webber,c Xiao-Hong Yuc and Roger J. Griffina,*
aNorthern Institute for Cancer Research, School of Natural Sciences-Chemistry, Bedson Building, University of Newcastle,
Newcastle Upon Tyne NE1 7RU, UK
bNorthern Institute for Cancer Research, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
cPfizer Global Research and Development/Agouron Pharmaceuticals Inc., 10770 Science Centre Drive, La Jolla, CA 92121, USA
Received 3 December 2003; revised 11 February 2004; accepted 5 March 2004
Abstract—The synthesis and biological evaluation of a new series of amine-substituted 2-arylbenzimidazole-4-carboxamide inhib-
itors of the DNA-repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is reported. The introduction of an amine substituent at
the 2-aryl position is not detrimental to activity, with most inhibitors exhibiting Ki values for PARP-1 inhibition in the low
nanomolar range. Two compounds in this series were found to potentiate the cytotoxicity of the DNA-methylating agent tem-
ozolomide by 4–5-fold in a human colorectal cancer cell line.
Ó 2004 Published by Elsevier Ltd.
1. Introduction
hydrolysis by poly(ADP-ribose) glycohydrolase.5
Depletion of NADþ, as a consequence of inappropriate
PARP-1 activity, results in critically low intracellular
ATP concentrations leading to extensive cell death. The
enzyme is thus implicated in the pathology of several
disease states, including diabetes, arthritis, and cardio-
vascular disease.6;7
The abundant eukaryotic nuclear enzyme poly(ADP-
ribose) polymerase-1 (PARP-1), is the founding member
of a burgeoning family of poly(ADP-ribosyl)ating
enzymes of diverse and complex function.1 A major role
of PARP-1 is in the base excision repair of DNA,
mediated by the ability of the enzyme to detect DNA
strand breaks.2–4 Utilising intracellular NADþ as a
substrate, PARP-1 catalyses the formation of long linear
and branched poly(ADP-ribose) polymers that modify a
variety of nuclear proteins, including histones (hetero-
modification), and the enzyme itself (automodification).
This is part of the immediate cellular response to various
forms of genotoxic stress, including DNA damage
caused by ionising radiation and DNA alkylating
agents. Poly(ADP-ribose) polymers have a short half
life, estimated to be less than 1 min, due to rapid
PARP-1 mediated DNA repair in response to DNA
damaging agents represents a mechanism of tumour
resistance, and inhibition of this enzyme has been shown
to enhance the activity of ionising radiation and several
cytotoxic antitumour agents, including temozolomide
and topotecan.8–10 PARP-1 is thus a potentially impor-
tant therapeutic target for enhancing DNA-damaging
cancer therapies. As part of a programme to develop
clinically useful inhibitors, we have employed a crystal
structure-based inhibitor design approach, utilising an
understanding of structure–activity relationships for
NADþ-competitive PARP-1 inhibition. These studies
have resulted in the identification of potent inhibitors,
including the benzimidazole-4-carboxamides (1),11 and
inhibitors that incorporate the essential carboxamide
motif within a tricyclic system (2 and 3).12;13
Keywords: PARP-1; Inhibitors; Benzimidazole-4-carboxamides; Che-
mopotentiation.
* Corresponding author. Tel./fax: +44-191-222-8591; e-mail: r.j.griffin@
ncl.ac.uk
0960-894X/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.03.017

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A. W. White et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2433–2437
Co-crystallisation of a 2-aryl-substituted benzimidazole-
4-carboxamide (1; R ¼ 3-OMe) with the C-terminal
catalytic domain of chicken PARP-1, revealed that the
2-aryl ring of the inhibitor occupied a large cavity within
the NADþ binding site.11 Importantly, this observation
suggested that substituents on the 2-aryl ring would be
tolerated without detriment to inhibitory activity. In this
communication we report the synthesis and biological
evaluation of a series of benzimidazole-4-carboxamides
bearing a range of amine groups on the 2-aryl ring.
These compounds were designed to probe the NADþ
binding pocket, with a view to enhancing potency and
modulating the pharmacokinetic and physicochemical
properties of this class of PARP-1 inhibitor.
sation of 5 with 2-(3-formylphenyl)-1,3-dioxolane15 in
DMA at 140 °C, in the presence of sodium hydrogen
sulfite, afforded the acetal derivative 6, which gave the
corresponding aldehyde 7 on acid hydrolysis.16 The
analogous reaction between 5 and terephthalaldehyde
mono(diethylacetal) gave aldehyde 8 directly, and
reduction of 7 and 8 with sodium borohydride furnished
the required alcohols 9 and 10, respectively. The target
amine-substituted 2-arylbenzimidazole-4-carboxamides
(13a–13f and 14a–14h) were prepared in variable yields
from 9 and 10, by conversion into the respective chlo-
rides 11 and 12 under Vilsmeier-like conditions (SOCl2-
DMF), followed by treatment with the appropriate
amine.17
O
O
O
N
NH
NH
NH2
3. Results and discussion
N
All new compounds were assayed for inhibition of
purified human PARP-1 as described previously,11 and
the results are presented in Table 1. The benchmark
PARP-1 inhibitor 3-aminobenzamide (3-AB), and the
HN
N
N
H
R
R
R
unsubstituted
2-phenylbenzimidazole-4-carboxamide
NU1070,11;18 are included for comparison. In general,
amine substitution at the 3- or 4-position of the 2-aryl
ring resulted in an enhancement of PARP-1 inhibitory
activity compared with NU1070, this effect being most
pronounced at the 4-position. The N-methylamino-
methyl (14a) and N,N-dimethylaminomethyl (13a and
14b) derivatives proved approximately 3-fold more
potent than NU1070, and while this activity was
retained with the 4-N,N-di-(2-hydroxyethyl)amino-
methyl analogue (14c), a dramatic reduction in potency
was observed when this group was introduced at the 3-
position (13b). The reason for this loss of activity
remains uncertain, but the high potency of the corre-
sponding piperidine and N-methylpiperazine derivatives
(13d and 13f) mitigates against unfavourable steric
interactions within the NADþ binding domain. Inter-
estingly, a morpholine function is also detrimental to
PARP-1 inhibitory activity when introduced at the 3-
position, but not the 4-position (compare 13e and 14f).
(1)
(2)
(3)
2. Chemical synthesis
Although we have previously reported the synthesis of a
wide range of benzimidazole-4-carboxamides, including
the key alcohols 9 and 10,11 an alternative synthetic
route was required to enable the preparation of these
intermediates on a multi-gram scale (Scheme 1). Cata-
lytic hydrogenation of 2-amino-3-nitrobenzamide (4),
prepared as described previously,11 afforded the unstable
anthranilamide 5 in quantitative yield, and this was used
directly in the next reaction step. The required benz-
imidazole-4-carboxamides were prepared from 5 using a
modification of a literature procedure.14 Thus, conden-
CONH2
NH2
CONH2
CONH2
CHO
R
R
R
N
N
iv
ii
N
H
N
H
X
(6); R = 3-CH[O(CH2)2O]
(4); X = NO2
(5); X = NH2
(9); R = 3-CH2OH
(10); R = 4-CH2OH
iii
i
(7); R = 3-CHO
(8); R = 4-CHO
CONH2
CONH2
R
R
N
N
v
vi
N
N
H
H
13a-13f; R = 3-CH2NR1R2
(11); R = 3-CH2Cl
(12); R = 4-CH2Cl
14a-14h; R = 4-CH2NR1R2
Scheme 1. Synthesis of benzimidazole-4-carboxamides. Reagents and yields: (i) 10% Pd/C, H2, MeOH, (100%); (ii) NaHSO3, DMA, 140 °C, (70–
80%); (iii) 0.5 M HCl, THF, (92%); (iv) NaBH4, H2O, EtOH, (94%); (v) SOCl2, DMF (cat.), MeCN, (93%); (vi) R1R2NH, (Et)3N, MeCN, (8–89%).

A. W. White et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2433–2437
Table 1. Inhibition of PARP-1 by amine-substituted 2-arylbenzimidazole-4-carboxamides
2435
CONH2
3
R
4
N
N
H
Compound no.
R
Ki (nM)a
Compound no.
R
Ki (nM)
3-ABb
NU1070
13a
––
H
3-CH2NMe2
3100
15
14a
14b
14c
4-CH2NHMe
4-CH2NMe2
4-CH2N(CH2CH2OH)2
3.2
4.5
4.5
5.6
13b
13c
3-CH2N(CH2CH2OH)2
272
14d
14e
5.9
7.2
N
N
4
4
10.2
N
3
O
N
13d
13e
11.7
31
14f
12
3
3
4
4
N
N
N
Me
O
N
14g
8.7
N
OMe
Me
Me
N
13f
9.8
14h
5.9
3
N
4
a Assayed against human PARP-1 full length protein; for further details see Ref. 11. Data are averages of at least two independent experiments,
standard deviation 610%.
b 3-Aminobenzamide.
Although cycloalkylamino substitution at the 3- or 4-
position did not increase potency to the same degree as
did the introduction of alkyl- or dialkyl-amines, the
differences are modest, and bulky amine substituents
appear to be well tolerated (compare 14a with 14h).
CH2NMe2) with the chicken PARP-1 catalytic
domain,12;13 an ionic interaction between Glu-763 (Gln-
763 in chicken PARP) and the protonated amine
substituent of inhibitors in the 2-arylbenzimidazole-4-
carboxamide series is also possible, and may account for
the increase in potency observed (Fig. 1).
These observations indicate that the increased potency
of this compound series compared with NU1070 may
arise through an additional binding interaction between
the pendant amine function and the large cavity within
the NADþ binding domain. Our previous studies with
tricyclic PARP-1 inhibitors bearing analogous amine
groups have demonstrated that unique changes in the
protein conformation occur on inhibitor binding.13
Notably, the co-crystal structure of the tricyclic indole
lactam (2, R ¼ 4-CH2NMe2) with the PARP-1 catalytic
domain, revealed that disruption of a key hydrogen
bond between residues Tyr-889 and Asp-766 is necessary
for the 4-dimethylaminomethyl group to be accommo-
dated in the large cavity, resulting in relocation of an
entire loop between Gly-888 and Gly-892. Given that
the benzimidazole-4-carboxamide (1) and tricyclic lac-
tam (2 and 3) pharmacophores exhibit near-identical
binding geometries and hydrogen bonding interactions,
it is likely that similar conformational changes to the
protein are elicited by the amine-substituted 2-aryl-
benzimidazole-4-carboxamides. Although not observed
in complexes of the tricyclic inhibitors 2 and 3 (R ¼ 4-
The activity of selected PARP-1 inhibitors as potenti-
ators of DNA-damaging antitumour agent cytotoxicity,
was assessed in vitro by treating human lung (A549)
Ser-904(OH)
Gly-863(NH)
Gly-863(CO)
O
N
H
N
H
N
H
R
N
H
H
O
3
Glu-988
(CO2H)
R1
H
4
Glu-763
-
(CO2
)
Tyr-889(OH)
Figure 1. Interactions of 2-arylbenzimidazole-4-carboxamides with the
PARP-1 NADþ binding domain, indicating the likely positioning of an
amine substituent within the cavity.12;13

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A. W. White et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2433–2437
Table 2. Potentiation of temozolomide (TM) and topotecan (TP)
activity by selected PARP-1 inhibitors in A549 and LoVo human
tumour cell linesa
125
100
75
50
25
0
Compound
no.b
PF50 (A549)c
TM
PF50 (LoVo)d
TP TM
TP
13a
13b
13c
14b
14c
14d
14e
ND
1.1
ND
1.9
1.3
1.8
1.9
ND
1.1
1.4 0.3
ND
1.7 0.1
ND
ND
1.6
1.1 0.1
1.7 0.4
ND
1.7 0.2
4.0 1.0
ND
ND
1.5
1.5 0.3
ND
5.2 1.9
ND
ND
a Cell growth was estimated by the SRB assay.20
b 0.4 lM inhibitor concentration.
c Data are mean of two independent experiments.
d Data are mean standard deviation of at least three independent
experiments.
0
250
500
750
1000
1250
1500
[Temozolomide]µM
Figure 2. Inhibition of LoVo cell growth by temozolomide (TM);
potentiation by 14b. Cells were exposed to increasing concentrations of
temozolomide alone (solid circles and line) or in the presence of 0.4 lM
14b (open circles, broken line) for five days. Data (normalised to no
drug or 0.4 lM 14b alone) are from a single representative experiment
in which the IC50 for temozolomide alone was 1006 lM, but 199 lM in
the presence of 14b, giving a PF50 of 5.1 in this experiment.
and/or colorectal (LoVo) cancer cell lines with increas-
ing concentrations of two antitumour drugs, the DNA
methylating agent temozolomide (TM) and the topo-
isomerase I inhibitor topotecan (TP), in the presence of
a fixed concentration of PARP-1 inhibitor (Table 2).
The cytotoxic potentiation factor (PF50) of these agents
was expressed as the ratio of the GI50 for cells treated
with cytotoxic agent and PARP-1 inhibitor, against the
GI50 of cells exposed to the cytotoxic drug alone (where
GI50 is the concentration of drug required to inhibit
growth by 50%). Thus a PF50 value of 1.0 indicates no
effect.9;19
with improved water solubility compared with NU1070,
when formulated as amine salts.
Acknowledgements
In general, the PARP-1 inhibitors exhibited a modest
potentiation of the activity of TM and TP in the A549
tumour cell line, with compounds 14b, 14d, and 14e
giving similar activity consistent with their PARP-1
inhibitory activity. Compound 13b, a much weaker
PARP-1 inhibitor, exhibited negligible potentiation, and
the lower PF50 value observed for 14c in combination
with TP, perhaps reflects the lower cell permeability
arising from the polar diethanolamino group. Although
a similar modest degree of potentiation of TP activity
was seen in the LoVo tumour cell line, in the presence of
PARP-1 inhibitors, a significant effect was observed with
TM in these cells. Thus, the cytotoxicity of TM was
increased 4-fold and 5-fold, respectively, by the N,N-
dimethylaminomethyl (14b) and pyrrolidinomethyl
(14d) derivatives, both of which are potent PARP-1
inhibitors. The results for compound 14b are also rep-
resented graphically in Figure 2.
The authors thank Cancer Research UK for financial
support.
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