Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase

Article abstract of DOI:10.1016/j.bmcl.2008.06.025

We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides were developed with restricted conformation throug

Full text of DOI:10.1016/j.bmcl.2008.06.025

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3942–3945
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase
*
Keith A. Menear , Claire Adcock, Francisco Cuenca Alonso, Kristel Blackburn, Louise Copsey,
Jan Drzewiecki, Alexandra Fundo, Armelle Le Gall, Sylvie Gomez, Hashim Javaid, Carlos Fenandez Lence,
Niall M. B. Martin, Chris Mydlowski, Graeme C. M. Smith
KuDOS Pharmaceuticals Ltd, 410 Cambridge Science Park, Milton Road, Cambridge CB4 0PE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 February 2008
Revised 6 June 2008
Accepted 6 June 2008
Available online 12 June 2008
We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted
benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides
were developed with restricted conformation through intramolecular hydrogen bond formation; the
compounds exhibit low nM enzyme and cellular activity as PARP-1 inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Poly(ADP-ribose) polymerase-1
PARP-1
PARP-2
The role of PARP-1 (poly(ADP-ribose) polymerase-1) has been
recognized as a key component in a number of cellular processes
including necrosis and apoptosis, transcriptional regulation and
DNA repair.1 Due to this, PARP-1 has been implicated in several
important disease states including cerebro-vascular disease, reper-
fusion injury and cancer resistance.2,3 PARP-1 is an abundant nu-
clear enzyme that binds to predominantly single stranded
damaged DNA, from where it becomes activated and leads to the
repair of the DNA via the base-excision repair (BER) pathway.1 Uti-
lizing nicotinamide adenine dinucleotide (NAD+) as substrate,
PARP-1 catalyzes the formation of ADP-ribose polymers through
either automodification of itself or ribosylation of other protein
acceptors that aid in the process of repair.
O
O
H
NH
N
N
H
O
1 PARP-1 IC50 0.77μM
Figure 1. Compound 1 PARP-1 IC50 0.77
l
M.
PARP-1 inhibition is known to potentiate the effects of radiation
as well as a variety of chemotherapeutic agents in tumour models.4
Moreover, recent studies have also indicated that PARP-1 inhibi-
tors may be used as potential stand-alone therapies where the tu-
mours have become deficient in certain DNA repair pathways.5,6
Tumours that have lost their BRCA1 and BRCA2 components of
the homologous recombination DNA repair pathways, for example,
show high sensitivity to the effects of PARP inhibition. This concept
of synthetic lethality along with the combination of PARP inhibi-
tors and radio/chemotherapies is now under investigation pre-
clinically and in the clinic.
mized compounds from the phthalazinone series are shown in Ta-
ble 1. Here, the 3-piperazine-1-carbonyl substituent 2 and acylated
derivatives 3 and 4 (tested as the racemate) demonstrate excellent
activity with PARP-1 IC50 values in the low nM range and cellular
potency, as measured by the potentiation factor PF50 to the methy-
lating agent methyl methanesulfonate (MMS), greater than 2. For
compound 4, a near maximal sensitization is seen (PF50 29.7) in
the absence of intrinsic cellular toxicity, which may indicate high
cellular permeability or differing kinetics of PARP inhibition.
In an effort to explore alternative chemical templates, we took
the expedient of dissecting the phthalazinone B ring and introduc-
ing an oxygen linker into the system (Fig. 1). The hypothesis for
this approach was that the subsequent intramolecular hydrogen
bond between the aryl C-2 oxygen and benzamide NH may counter
a loss in entropy in the acyclic system. Additionally this arrange-
ment will conformationally lock the system in the required syn
configuration necessary for bidentate hydrogen bonding to the
We have previously reported a series of substituted benzyl-
phthalazinones originating from compound 1 (Fig. 1) as potent,
cellularly active inhibitors of PARP-1.7,8 Typical examples of opti-
* Corresponding author. Tel.: +44 1403 248844; fax: +44 1403 248855.
E-mail address: kmenear@kudospharma.co.uk (K.A. Menear).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.025

K. A. Menear et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3942–3945
3943
Table 1
Table 2
Structure–activity relationships of substituted 2-benzyloxybenzamides
O
O
NH
N
NH2
O
O
O
N
N
N
R1
N
R2
R1
a
Compound
R1
H
PARP IC50
0.002
(
lM)
PF50
Compound
R1
R2
PARP-1 IC50
(l
M)
PF50
2
2.7
4.8
5
6
H
H
H
F
0.018
0.061
1.3
1.4
O
O
3
0.008
O
7
H
F
0.188
0.024
0.036
0.050
1.1
O
4
0.005
29.7
O
O
8
1.96
2.0
a
The PF50 value is the potentiation factor, calculated as the ratio of the GI50
growth curve for the alkylating agent, methyl methane sulfonate (MMS) divided by
the GI50 of the curve of MMS + PARP inhibitor. The cells used were HeLa B and the
test compounds were used at a fixed concentration of 200 nM. PARP-1 IC50 and PF50
values are means from 2 to 3 independent dose–response curves.
O
O
9
H
F
O
10
3.7
enzyme (carbonyl of Ser904 and NH of Gly863) characteristic of
this class of inhibitor.9
A similar tactic has been described
previously for benzamidazole carboxamides; however, the ap-
proach was detrimental to potency in comparison to equivalent
bonded ring systems.10,11
Table 3
A further consideration for developing the alkoxybenzamide
template was ready chemical access to explore SAR, which might
otherwise have proven difficult with the phthalazinone class. The
assumption, however, was that SAR between the phthalazinone
series and the putative alkoxybenzamide scaffold would be trans-
ferable. To establish this, matched pairs of compounds with the
equivalent substituted phthalazinones 2, 3 and 4 were initially
proposed for synthesis and testing.
Structure–activity relationships of substituted 2-phenethyloxybenzamides
O
R2
R1
NH2
N
N
O
O
Compound
R1
R2
PARP-1 IC50
(l
M)
PF50
Gratifyingly, the benchmark 2-[3-(piperazine-1-carbonyl)ben-
zyloxy]benzamide 5 (Table 2) had excellent inhibitory potency
against PARP-1 with an IC50 of 18 nM. This initial compound there-
fore proved the equivalence of the two chemical templates to-
wards PARP-1 inhibition. Likewise the cyclopentanecarbonyl 7
and the phenoxypropionylcarbonyl 9 compounds were made, but
in these cases, although still potent, a loss of relative activity was
observed. All three compounds were tested in cellular assays to
establish their effect on the potentiation of cytotoxicity to MMS.
For compounds 5 and 7, little effect was seen; however, 9 gave
good cellular activity with a PF50 of 2.0. We have previously ob-
served that for the phthalazinone series cellular activity can be en-
hanced by 4-fluoro substitution on the benzyl linker.7,8 This trend
is also noted for the alkoxybenzamides with the corresponding 4-
fluoro analogues 6, 8 and 10 all showing improved PF50 values over
the des-fluoro derivatives (5, 7 and 9). Indeed, the phenoxypropio-
nyl 10 proved to be the most cellularly active compound from the
series with a PF50 of 3.7.
11
12
H
H
H
F
0.97
>10
1.06
ND
O
13
14
15
16
H
F
0.2
ND
ND
ND
1.5
O
O
0.103
0.2
O
H
F
O
O
0.019
As a further exploration of the SAR around the pendant ether
linker, a series of phenethyloxybenzamides were synthesized,
again maintaining the three benchmark side chains for comparison
(Table 3). Homologation of the linker by one carbon atom to the
simple 3-piperazine carbonyl 11 results in a dramatic reduction
in PARP-1 potency (over 50Â). The decline in potency was also ob-
served for 15 (vs 9) but not, interestingly, for 13 which is equipo-
tent to 7. Again the tactic of introducing a 4-fluoro-benzyl
substituent in order to improve enzyme and cell potency was at-
tempted and gave varying degrees of success. For the 3-piperazine
carbonyl 12 derivative activity was markedly reduced, whereas for
14, and to a greater extent for 16, activity was enhanced. Curiously
for 16, the potency against PARP-1 was improved over the corre-
sponding benzyl-linked compound 10 (IC50 19 nM vs 50 nM,
respectively). The reason for this paradox is uncertain but the
SAR suggests alternative PARP-1 binding modes for the differing
sub-series of compounds.
Our attention next turned to an exploration of substituents on
the benzamide ring. An examination of the homology model of

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K. A. Menear et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3942–3945
Table 4
in this class has limited room for substitution especially at C4–6
(R1–3 in Table 4).
Structure–activity relationships of substituted 2-benzyloxyfluorobenzamides
R1
O
With this in consideration and also from our experience show-
ing that an aryl fluorine substitution at C-4 of the benzyloxy linker
gives enhanced potency, the chemistry was restricted to variously
decorated fluorobenzamides (Table 4). To this end, compounds
17–21 were synthesized. Of greatest value was the observation
that the 5-fluoro-2-alkoxy benzamide 17 had good potency against
the enzyme in excess (by over threefold) of the C-5 des-fluoro
derivative 6. Table 4 shows that a significant reduction in potency
results from the other exemplified patterns of fluoro substitution
with the difluoro analogue 21 in particular being significantly less
R2
NH2
R3
O
O
F
R4
N
NH
Compound
R1
R2
R3
R4
PARP-1 IC50
(l
M)
PF50
17
18
19
20
21
H
H
H
H
F
F
F
H
F
H
H
F
F
H
H
H
H
H
F
0.029
0.24
0.25
1.5
1.6
1.08
1.2
0.9
ND
active with an IC50 > 10 lM.
In an attempt to determine whether the C-5 fluoro-benzamide
in 17 would function additively with the capped piperazine side
chains, the adducts 22 and 23 were synthesized (Table 5). In both
cases potency was improved in comparison to the C-5 des-fluoro
analogues 8 and 10. On going from 8 to 22 IC50 enhanced by 2Â,
on going from 10 to 23 IC50 enhanced by 4Â. Disappointingly,
the potency advantage at the level of the enzyme was not reflected
in an increase in cellular activity where only modest values were
noted.
F
>10
Table 5
Structure–activity relationships of difluoro-substituted 2-benzyloxybenzamides
O
F
NH2
For the analogues described above, synthetic access was readily
achieved through ether formation of an appropriately substituted
hydroxylbenzamide 24 (Scheme 1). Two standard methods were
employed to achieve this result; for the benzyl-substituted com-
pounds, classical Williamson ether formation with the requisite
benzyl bromide methyl ester (either X = F or H) gives intermediate
25. Alternatively, Mitsunobu reaction with either an appropriately
substituted benzyl alcohol or substituted 2-phenylethanol using
diisopropyl azodicarboxylate (DIAD) gave 25 where Cn, n = 1 or 2,
respectively. For the extended linker (Cn, n = 2, compounds 11–
16) this was the method of choice. Simple base hydrolysis of the
methyl ester 25 to the carboxylic acid 26, using methanolic sodium
hydroxide, followed. Coupling of 26 with BOC-piperazine under
standard conditions furnished 27, which underwent deprotection
to the free amine 28 under acidic conditions. Where necessary,
capping of the piperazine side chain was conducted under basic
conditions with either cyclopentane carbonyl chloride or 2-phen-
oxy propionyl chloride to give intermediates 29.
O
O
F
N
N
R1
Compound
R1
PARP-1 IC50
0.012
(l
M)
PF50
1.5
O
O
22
O
23
0.014
1.12
PARP-1 (previously described) and co-crystallized inhibitors from
other workers11 makes it clear that the binding pose for inhibitors
O
O
O
O
i or ii
iii
NH2
NH2
NH2
Y
Y
Y
O
O
X
O
X
OH
(C)n1,2
(C)n1,2
OH
OMe
24
25
X=H or F
26
iv
O
O
O
O
NH2
NH2
Y
vi
v
Y
NH2
Y
O
O
X
O
O
X
O
(C)n1,2
(C)n1,2
N
N
(C)n1,2
N
N
N
R
H
N
O
X
O
29
28
27
Scheme 1. Reagents and conditions: (i) substituted benzyl bromide, acetone, K2CO3, rt; (ii) substituted benzyl alcohol or substituted 2-phenylethanol, acetone, Ph3P, DIAD, rt;
(iii) NaOH, MeOH, rt; (iv) DMF, HBTU, DIPEA, tert-butyl piperazine-1-carboxylate; (v) concd HCl, EtOH, rt; (vi) DCM, DIPEA, ROCl.

K. A. Menear et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3942–3945
3945
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series of benzyl-phthalazinones. Further evaluation of this class
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8. Cockcroft, X.; Dillon, K. J.; Dixon, L.; Drzewiecki, J.; Loh, V. M.; Kerrigan, F.;
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