4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl] -2H-phthalazin-1-one: A novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1

Article abstract of DOI:10.1021/jm8001263

Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-, or ionizing radiation-induced DNA damage and represents a new target for cancer therapy. In this article, we disclose a novel series of substituted 4-benzyl-2H-phthalazin-1-ones that possess high inhibitory enzyme and cellular potency for both PARP-1 and PARP-2. Optimized compounds from the series also demonstrate good pharmacokinetic profiles, oral bioavailability, and activity in vivo in an SW620 colorectal cancer xenograft model. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl] -2H-phthalazin-1-one (KU-0059436, AZD2281) 47 is a single digit nanomolar inhibitor of both PARP-1 and PARP-2 that shows standalone activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently undergoing clinical development for the treatment of BRCA1- and BRCA2-defective cancers.

Full text of DOI:10.1021/jm8001263

J. Med. Chem. 2008, 51, 6581–6591
6581
4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: A
Novel Bioavailable Inhibitor of Poly(ADP-ribose) Polymerase-1
Keith A. Menear,*,† Claire Adcock,† Robert Boulter,† Xiao-ling Cockcroft,† Louise Copsey,† Aaron Cranston,†
Krystyna J. Dillon,† Jan Drzewiecki,† Sheila Garman,† Sylvie Gomez,† Hashim Javaid,† Frank Kerrigan,‡ Charlotte Knights,†
Alan Lau,† Vincent M. Loh, Jr.,† Ian T. W. Matthews,† Stephen Moore,† Mark J. O’Connor,† Graeme C. M. Smith,†
and Niall M. B. Martin*,†
KuDOS Pharmaceuticals Ltd., 410 Cambridge Science Park, Milton Road, Cambridge, U.K. CB4 0PE, and Thermo Fisher Scientific Ltd.,
TreVillett, Tintagel, Cornwall, U.K. PL34 0HW
ReceiVed February 6, 2008
Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-, or ionizing
radiation-induced DNA damage and represents a new target for cancer therapy. In this article, we disclose
a novel series of substituted 4-benzyl-2H-phthalazin-1-ones that possess high inhibitory enzyme and cellular
potency for both PARP-1 and PARP-2. Optimized compounds from the series also demonstrate good
pharmacokinetic profiles, oral bioavailability, and activity in vivo in an SW620 colorectal cancer xenograft
model. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one (KU-
0059436, AZD2281) 47 is a single digit nanomolar inhibitor of both PARP-1 and PARP-2 that shows
standalone activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently undergoing
clinical development for the treatment of BRCA1- and BRCA2-defective cancers.
Introduction
it utilizes NAD+ to synthesize poly(ADP)ribose chains either
on itself (poly(ADP)ribosylation) or on a variety of nuclear
target proteins, including topoisomerases, histones, and p53.1,10
This process of PARP-1 binding and activation helps amplify
the repair process that is known as base excision repair (BER)
in which SSBs are targeted. SSBs are generally introduced into
the DNA by oxidative damage that are caused by the cell’s own
metabolic processes as well as by exogenous chemical damage
or ionizing radiation. PARP-1 knockout (PARP -/-) cells and
animals exhibit high sensitivity when they are exposed to
alkylating agents and γ irradiation.11,12 Moreover, in studies
that use low-molecular-weight inhibitors of PARP-1, it has been
shown that the effects of radiation are enhanced by the
suppression of the repair of potentially lethal DNA damage.13,14
In cells that are treated with alkylating agents, the inhibition of
PARP-1 leads to a marked increase in DNA strand breakage
and cell killing.15,16 The use of more recent selective and potent
PARP-1 inhibitors has confirmed that the inhibition of PARP-1
activity potentiates a wide variety of important cancer cytotoxins
including 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetra-
zine-8-carboxamide (temozolomide (TMZ)), tecans such as (S)-
10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3,4:
6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione (topotecan),
and cis-diamminedichloroplatinum(II) (cisplatin).17-21
Poly(ADP-ribose) polymerase-1, or PARP-1a, is a 113 kDa
nuclear protein that is involved in the signaling of DNA damage
through its ability to recognize and rapidly bind DNA single-
or double-strand breaks.1 PARP-1 is also known to participate
in a variety of cellular functions, including gene amplification
and transcriptional regulation, cell division, and differentiation
as well as apoptosis and chromosome stability.2,3 PARP-1 is
the most abundant protein of a family of some 18 proteins that
are now identified.4,5 Its closest homolog is PARP-2, which is
∼62% identical in its catalytic domain to PARP-1, and unlike
the rest of the PARP family, it also has the ability to bind DNA
and compensate for PARP-1 activity in DNA single-strand break
(SSB) repair.6 Other PARP superfamily members include vault
PARP, which participates in macroparticle excretion through
its inclusion in vault bodies, and the more extensively studied
polyribosylating protein tankyrase (of which there are two
distinct proteins), which helps regulate telomerase activity at
the chromosomal telomere tails.7,8
Studies on the mechanism by which PARP-1 modulates DNA
repair and other processes have identified its ability to produce
long chains of poly(ADP-ribose) (PAR) within the cell nucleus
as being central to its activity.9 Following DNA damage,
PARP-1 is rapidly activated by binding to DNA breaks, where
The potential of PARP inhibition within oncology was further
highlighted by reports that cell lines deficient in BRCA1 and
BRCA2 (proteins involved in homologous recombination (HR)
repair22) are highly sensitive to PARP-1 inhibitors resulting in
cell death.23,24 BRCA1 and BRCA2 are known tumor suppressor
genes whose wild-type alleles are frequently lost in tumors of
heterozygous carriers.25,26 The association of BRCA1 and
BRCA2 protein mutations with breast cancer is well character-
ized.27 Carriers of mutations in BRCA1 and BRCA2 are also at
an elevated risk of cancer of the ovary, prostate, and pancreas.
Therefore, the possibility exists that PARP-1 inhibitors will be
of high therapeutic benefit for tumors that are deficient in these
specific proteins and potentially for other DNA-repair HR
proteins that may be lost within the tumorigenesis process.28-30
* To whom correspondence should be addressed. (K.A.M.) E-mail:
kmenear@kudospharma.co.uk. (N.M.B.M.) Tel: (44) 1223 719 719. Fax:
(44) 1223 719 720. E-mail: nmartin@kudospharma.co.uk.
†
KuDOS Pharmaceuticals Ltd.
Thermo Fisher Scientific Ltd.
‡
a Abbreviations: PARP, poly(ADP-ribose)polymerase; SSB, single-strand
break; BER, base excision repair; DSB, double-strand break; HR, homolo-
gous recombination; MMS, methyl methanesulfonate; PAR, poly(ADP-
ribose); TMZ, temozolomide; RTV, relative tumor volumes; Boc, tert-
butoxycarbonyl; DMSO, dimethylsulfoxide; TFA, trifluoroacetic acid; DCM,
dichloromethane; DMA, dimethyl acetamide; DMF, dimethyl formamide;
ether, diethyl ether; Et3N, triethylamine; EtOH, ethanol; HCl, hydrochloric
acid; h, hours; IPA, isopropyl alcohol; THF, tetrahydrofuran; DIPEA, N′N′-
diisopropylethylamine; NH3OH, ammonium hydroxide; MeOH, methanol;
NaOH, sodium hydroxide; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tet-
ramethyluronium hexafluorophosphate.
10.1021/jm8001263 CCC: $40.75
2008 American Chemical Society
Published on Web 09/19/2008

6582 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Table 1. PARP-1 and Cellular Potencies of the Homopiperazine Series
Menear et al.
Table 2. PARP-1 and Cellular Potencies of Piperazine Series 23-38
a
compd
R1
R2
PARP-1 IC50 (µM)
PF50
compd
R1
R2
PARP-1 IC50 (µM)
PF50
14
15
16
17
18
19
20
21
22
H
F
H
F
H
H
H
F
0.007
0.018
0.007
0.018
0.023
0.020
0.008
0.012
0.012
12.6
5.4
18.0
2.2
1.4
1.4
2.5
3.7
4.2
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
F
H
F
H
F
H
F
H
F
H
F
H
F
H
F
0.002
0.005
0.010
0.002
0.002
0.003
0.012
0.008
0.008
0.006
0.051
0.005
0.011
0.006
0.039
0.012
3.7
8.6
2-hydroxyethyl
2-hydroxyethyl
benzyl
ethylsulphonyl
morpholine-4-carbonyl
morpholine-4-carbonyl
acetyl
2-pyridyl
2-pyridyl
24.3
>40a
>40a
>40a
4.6
4.9
2.3
8.4
ND
5.2
2-pyrimidinyl
2-pyrimidinyl
ethylsulfonyl
ethylsulfonyl
morpholine-4-carbonyl
morpholine-4-carbonyl
phenyl
phenyl
benzoyl
benzoyl
2-hydroxyethyl
2-hydroxyethyl
F
F
cyclopropanecarbonyl
a The PF50 value is the potentiation factor and is calculated as the ratio
of the IC50 growth curve for the alkylating agent methyl methanesulphonate
(MMS) divided by the IC50 of the curve of MMS + PARP inhibitor
(Experimental Section). The cells used were HeLaB, and the test compounds
were used at a fixed concentration of 200 nM. PARP-1 IC50 and PF50 values
are means from two to three independent dose-response curves. Variation
was generally (1% for PARP-1 and (15% for PF50.
7.2
8.2
1.8
3.3
a A PF50 value of >40 is indicative of a compound that causes complete
sensitization to MMS at a fixed concentration of 200 nM.
PARP activity was initially described over 40 years ago, and
since then there has been significant activity in the search for
pharmaceutically relevant inhibitors.3,31 However the correct
selection of inhibitor and clinical context has proven to be
difficult. To date, there are no drugs that modulate the target
that have reached the market, although a number of preclinical
and clinical agents are now being actively pursued, particularly
in cancer in combination therapy with established DNA-
damaging agents.31 These include Pfizer and the University of
Newcastle’s 8-fluoro-2-(4-methylaminomethylphenyl)-1,3,4,5-
tetrahydroazepino[5,4,3-cd]indol-6-one (AG014699)32 and In-
otek Pharmaceuticals’ undisclosed INO-1001,33 both for intra-
venous administration, and Abbott’s orally available 2-[(R)-2-
methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide (ABT-
888).34
4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluo-
robenzyl]-2H-phthalazin-1-one (47) is a novel, potent, and orally
bioavailable inhibitor of PARP-1 that has demonstrated exciting
clinical efficacy as a monotherapy in BRCA1- and BRCA2-
deficient ovarian and breast cancers.35 It is also under clinical
investigation in combination with DNA-damaging agents in-
cluding 5-(3,3,-dimethyl-1-triazenyl)-1H-imidazole-4-carboxa-
mide (dacarbazine) and cis-diamine(1,1-cyclobutanedicarbox-
ylato)platinum(II) (carboplatin). The following discussion
describes the potency refinement and the pharmacokinetic (PK)
optimization of a series of 4-benzyl-2H-phthalazin-1-ones
leading to the identification of 47.
acid intermediate 7. For the corresponding fluoro analog 8, in
which the fluoro that is ortho to the nitrile group is labile to
nucleophilic displacement, an alternative milder methodology
starting from 3-dimethoxyphosphosphoryl-3H-2-benzofuran-1-
one 4 was required. As such, the commercially available
phosphonate 4 was coupled to 2-fluoro-5-formylbenzonitrile 5
under standard conditions to produce 2-fluoro-5(3-oxo-3H-
isobenzofuran-1-ylidenemethyl)benzonitrile 6. A mixture of E
and Z isomers are isolated without separation and were treated
with hydrazine hydrate to produce the phthalazinone core. Base
hydrolysis of the pendant nitrile provides the second key
carboxylic acid intermediate 8.
With the fluoro and des-fluoro carboxylic phthalazinones in
hand, we established the chemistry to enable small focused
libraries to be generated by one of two methods (Scheme 2).
For a number of the piperazine and homopiperazine analogs,
the appropriately functionalized amine was coupled to the acid
with HBTU to provide reliably high yields of the target amides.
Alternatively, fluoro or des-fluoro carboxylic phthalazionones
7 or 8 were reacted with either Boc-piperazine or Boc-
homopiperazine to produce amides 9-12. Deprotection under
acid conditions yielded either the piperazine or homopiperazine-
carbonyl-benzylphthalazinone 10a, 14, 23, or 24, which were
suitable for further elaboration by parallel synthesis. As such,
standard conditions were employed in providing the alkyl, acyl,
aryl, sulfonamide, and urea derivatives that are described in
Tables 1-4.
Chemistry
The synthesis of both the piperazine and the homopiperazine
analogs that are described in Tables 1-4 was achieved via amide
bond coupling of the corresponding 3-[(4-oxo-3H-phthalazin-
1-yl)methyl]benzoic acid 7 or 2-fluoro-5-[(4-oxo-3H-phthalazin-
1-yl)methyl]benzoic acid 8 (Scheme 1). The synthesis of
corresponding intermediate 7 is outlined in Scheme 1. The
nucleophilic addition of 3H-isobenzofuran-1-one 1 to 3-formyl-
benzonitrile 2 in the presence of sodium methoxide provided
3-(1,3-dioxoinden-2-yl)benzonitrile 3 in good yield. The reaction
of indanone 3 with hydrazine hydrate provided the primary
phthalazinone core that, after base hydrolysis, furnished the key
Results and Discussion
Previously, we and others have described the identification,
synthesis, and optimization of a number of potent inhibitors of
PARP-1 that bear a common phthalazinone core.36-40 The focus
of these efforts was to optimize hits, which originated from a
medium throughput screen, that identified benzyl phthalazinone
13 as a moderately potent (IC50 ) 0.77 µM) PARP-1 antagonist
(Figure 1). A liability of the early compounds in the series was
a lack of cellular activity, which was measured by their ability
to sensitize HeLa B cells to the killing effect of the alkylating

NoVel BioaVailable Inhibitor of PARP-1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6583
Scheme 1. Synthesis Route for Phthalazinone Acid Intermediates 7 and 8a
a (i) Ethyl propionate, NaOMe, MeOH, reflux; (ii) Et3N, THF; RT; (iii) (a) NaOH(aq), THF, 72 h, 100 °C and (b) HCl (2 N); and (iv) NH2NH2 ·H2O, 3 h,
reflux.
Scheme 2. Synthesis Route for Phthalazinone Piperazine and Homopiperazine Analogsa
a (i) HBTU, substituted (homo)piperazine, DIPEA, DCM; (ii) HBTU, tert-butyl (homo)piperazine-1-carboxylate, DIPEA, DMA; (iii) 6 N HCl, EtOH;
and (iv) ROCl or RSO2Cl or RNCO, DIPEA, DCM or RCO2H, HBTU, DIPEA or NaBH4, RCHO.
agent methyl methanesulfonate (MMS). This issue was resolved
by the optimization of the pendant benzyl linker to provide
potent inhibitors of PARP-1 at both the enzyme and the cellular
level. In addition, the intrinsic pharmacokinetics of the series
were improved to yield good in vitro and in vivo half lives,
which culminated in the disclosure of 4-[[3-(1,4-diazepane-1-
carbonyl)-4-fluorophenyl]methyl]-2H-phthalazin-1-one 14.
The focus of our efforts that lead to the identification of
homopiperazine 14 was to provide a clinically useful PARP-1
inhibitor for use in combination with known cytotoxic agents
following iv administration. However, we reasoned that clinical
utility and patient compliance would be significantly enhanced
by the development of an orally available inhibitor of PARP-1
that would be suitable for longer-term treatments as a mono-
therapy or in combination. As the subject of this article, our
efforts were concentrated on the introduction of oral bioavail-
ability to the benzylphthalazinone series.
To reach this objective, we established the chemistry to afford
ready access to a number of designed focused libraries. Not all
data are represented here, and only compounds that are
illustrative of the key structure-activity relationships (SARs)
are discussed. From the activities described below, over 1000
compounds were generated, 87 and 43% of which had PARP-1
IC50 values that were <50 and <10 nM, respectively, and 53%
that had a PF50 value of >2. Throughout the library design
process, consideration was given to the key physicochemical
properties that might influence oral absorption. In particular,
we made efforts to limit the molecular weight and the polar

6584 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Menear et al.
Figure 1. Structures of 4-benzyl-2H-phthalazin-1-one 13, homopiperazine analog 14, and clinical candidate 47.
Table 3. PARP-1 and Cellular Potencies of Alkylpiperazines 39-44
surface area (<550 and <140 Å2, respectively) together with
the number of rotatable bonds (<7), the total number of
hydrogen-bond donors/acceptors (<10), the lipophilicity, and
the pKa (basic <8) while continuing to maintain the necessary
solubility (>0.1 mg/mL).41
We reasoned that a key impediment to oral availability in 14
was the high pKa (calculated value of 9.87) of the distal nitrogen
on the homopiperazine side chain. Previous investigators have
designed inhibitors of PARP-1 to mimic the substrate-protein
interactions of NAD+ with the enzyme.17 Mechanistically, these
compounds inhibit PARP-1 by blocking the binding of the
substrate, particularly the nicotinamide moiety, to the active site.
Early weak inhibitors such as 3-aminobenzamide42 have been
developed into more potent PARP-1 inhibitors that are derived
from a range of related pharmacophoric templates. Through a
previously developed SAR, we reasoned that compound 14
similarly binds into the nicotinamide substrate binding pocket
of the active site of PARP-1 and makes a key bidentate
interaction with Ser904 and Gly863, which is characteristic of
this class of pharmacophore. Additionally, this binding pose
suggests that the functionalization of the homopiperazine is well
tolerated without detrimental effects on enzyme activity. This
makes it possible to modulate the physicochemical properties
of compound 14 by the appropriate substitution at the distal
nitrogen.
To this effect, 2-hydroxethyl derivatives 15 and 16 were
synthesized to induce a reduction in the pKa of the basic nitrogen
via the ꢀ effect of the side-chain oxygen (Table 1). Typically,
analogs were made in both the 4-fluoro and des-fluorobenzyl
series for comparison. The resulting compounds 15 and 16
illustrate that alkylation at N4 is tolerated with the preservation
of good enzyme and cell potency. The N4 benzyl-substituted
compound 17 continues to maintain PARP activity, albeit with
only modest cellular potency. The removal of the entire basic
center with the sulfonamide and urea derivatives 18, 19, and
20 demonstrated that the cationic center is not a prerequisite
for activity although cellular potency is reduced vis-a-vis
compound 14. Likewise, acyl adducts 21 and 22 maintain good
PARP-1 activity but are similarly compromised against cell
activity compared to 14, which is possibly due to limited cellular
permeability.
compd
R1
R2
PARP-1 IC50 (µM)
PF50
24
39
40
41
42
43
44
H
F
H
F
F
F
F
F
0.005
0.088
0.013
0.006
0.006
0.005
0.004
8.6
1.02
2.5
7.7
7.3
6.4
6.5
methyl
methyl
ethyl
n-propyl
n-butyl
cyclopropylmethyl
fluoro series furnished the 2-pyridyl and 2-pyrimidinyl analogs
25-28, respectively. Both substitutions gave highly potent
PARP-1 inhibitors (IC50 < 5 nM) with significant improvements
in cellular activity over the matched fluoro compound 24 and
unsubstituted compound 23. Ethylsulfonamide 29 was more
successful in the piperazine series (cf. homopiperazine sulfona-
mide 18), which was also true for urea derivatives 31 and 32.
A loss of enzyme potency is observed when R1 is phenyl and
R2 is hydrogen (compound 33), although this is regained 10-
fold when R2 is replaced by fluorine (compound 34). Benzoyl
compounds 35 and 36 show that the enzyme can tolerate
reasonably large functionality while maintaining potency al-
though entries 37 and 38, corresponding to the homopiperazine
2-hydroxyethyl analogs 15 and 16, show a reduction in absolute
PF50 values (i.e., PF50 ) 18 and 3.3 for compounds 16 and 38,
respectively).
The influence of the 4-fluorine substitution has been previ-
ously noted, but is particularly evident in this series. A matched-
pair analysis shows a mean PF50 difference of 2.67, which is a
significant enhancement over that of the 4-hydrogen. The similar
enzyme potencies suggest that the introduction of a fluorine at
C-4 on the benzyl linker enhances the permeability. Interestingly,
this does not appear to be a global change in molecular
properties because the introduction of a fluorine at positions
other than the benzyl C-4 (data not shown) does not replicate
this effect. A possibility may be that repulsive dipole-dipole
interactions with the pendant C-3 carbonyl may restrict con-
formational rotation and molecular entropy, which leads to a
more permeable compound.
In a logical extension to the homopiperazine series, a number
of corresponding piperazine analogs were explored (Table 2).
Interestingly, in the direct comparison with homopiperazine 14,
compound 24 is equipotent for PARP-1 but is marginally less
active in the cellular sensitization assay (PF50 ) 12.6 vs 8.6,
respectively), which may be indicative of a reduction in cell
permeability. However, as anticipated, the side-chain SARs are
broadly superimposable. Again, the tactic of N4 substitution
with piperazine side chains that are designed to modulate
permeability/absorption characteristics was employed. The at-
tachment of heterocyclic groups in both the fluoro and des-
Beyond this observation, convincing structure-PF50 relation-
ships have been elusive within the phthalazinone series. Modest
changes to structure can have unanticipated effects on PF50
values, and, despite the analysis of a large data set, correlations
of PF50 with permeability parameters such as lipophilicity and
solubility have been disappointing. A set of piperazine deriva-

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6585
Table 4. PARP-1 and Cellular Potencies of Acylpiperazines 45-51
Table 6. Pharmacokinetic Screen of Representative Compounds in
Mousea
plasma concn (µg/mL)
compd
30 min
60 min
27
34
46
47
50
51
0.30
0.70
0.67
0.23
0.28
0.16
0.14
0.31
0.30
0.59
0.13
0.15
a Compound dosed po at 10 mg/kg in vehicle 10% DMSO/10%
cyclodextrin in PBS.
compd
R1
PARP-1 IC50 (µM)
PF50
45
46
47
48
49
50
51
methyl
ethyl
0.006
0.005
0.005
0.002
0.015
0.003
0.002
7.97
24.4
25.8
11.8
11.1
39.4
29.8
Table 7. Pharmacokinetics of Compound 46 in Rat and Compound 47
in Rat and Dog
cyclopropyl
cyclopentyl
cyclohexyl
2-tetrahydrofuryl
3-tetrahydrofuryl
46
47
compd
T1/2 (min)
Cl (mL/min/kg)
AUC0-inf (µg/mL/min)
Vd (L/kg)
rata
rata
dogb
55
53
49
304
1.18
100
170
5.4
511
1.3
35.4
423
0.91
40
Table 5. IC50 Measurements of Compound 47 against PARP-1,
PARP-2, and Tankyrase-1
enzyme
IC50 (µM)a
BAV (%)
100
a Compound dose for rat: iv and po at 15 mg/kg (10% DMSO/10%
cyclodextrin in PBS). b Compound dose for dog: iv at 2.5 mg/kg (10%
DMSO/10% cyclodextrin in PBS) and po at 10 mg/kg (methylcellulose
vehicle).
PARP-1
PARP-2
tankyrase-1
0.005
0.001
1.5
a Values are means from two to three independent dose-response curves.
Variation was generally (1% for PARP-1 and -2 and (15% for tankyrase-
1.
which illustrates that by the use of this method compounds can
be ranked in relative terms to provide an indication of oral
exposure. A doubling in exposure at 60 min is seen for N-phenyl
34, which is over and above that for 2-pyrimidinyl 27, although
poor solubility (0.001 mg/mL) prevented the further advance-
ment of this compound. In contrast, 2- and 3-tetrahydrofuryla-
mides 50 and 51 had enhanced water solubility, but their
corresponding exposure levels were relatively poor. The cyclo-
propylamide, compound 47, showed the greatest levels of
absorption by the use of this limited time-point assay. This
compound, along with ethylamide compound 46, which also
showed good potency in enzyme-free and cellular PF50 assays,
was advanced to further PK analysis.
tives that did show transferable PF50 profiles is illustrated in
Table 3. Here simple alkyl homologation provides compounds
with a significant increase in cellular activity from C-1 to C-2
elongation (compounds 39-41). The SAR for the series shows
an optimum cellular activity that decreases as lipophilicity
increases (compounds 42-44), and within this compound set,
only ethyl 41 (PF50 ) 7.7) approaches the activity of simple
unsubstituted compound 24. The pattern of activity that is
represented by 39 to 41 in Table 3 was transferable to an
alternative amide series that is outlined in Table 4. Acylation
of 24 to produce 4-acetyl piperazine 45 provided a compound
with good enzyme and cell-kill enhancement. As previously
described for the alkyl-capped piperazines (Table 3), the fine-
tuning of the R1 aliphatic side chain to ethyl 46, cyclopropyl
47, cyclopentyl 48, and cyclohexyl 49 gave excellent enhance-
ment in PF50 over acetyl alone. Furthermore, the introduction
of a 2- or 3-tetrahydrofurylamide group (50 and 51, respectively)
enhances both PF50 and aqueous solubility (0.98 mg/mL for 50).
It is interesting that there is a significant enhancement in both
enzyme and cell potency for cyclopropylamide 47 in comparison
with that for its homopiperazine counterpart 22 (IC50 ) 12 vs
5 nM and PF50 ) 4.2 vs 25.8, respectively).
The selectivity profile of 47 is typical of the inhibitors tested
in this class (Table 5). As expected from the close homology
between PARP-1 and PARP-2, 47 is essentially equipotent
against the two PARP isoforms, whereas it is 300 times less
effective against the inhibition of tankyrase.
As had been alluded to earlier, the discrimination of
compounds on the basis of potency or selectivity alone was
problematic because of the high number of actives that were
generated. Therefore, in an effort to address the objective of
finding an orally bioavailable PARP-1 inhibitor, we utilized a
medium-throughput mouse in vivo pharmacokinetic assay.
Typically, inhibitors were administered both iv (data not shown)
and po. Samples were collected over only limited time points,
and inhibitor plasma levels at 30 and 60 min were predominantly
used for ranking. Representative data are provided in Table 6,
Pharmacokinetic Profiles
To establish whether one of these compounds (46 or 47) had
the right PK profile to warrant its selection as an orally
bioavailable candidate for clinical use, we initially evaluated
both compounds for oral bioavailability and pharmacokinetic
stability in rats (Table 7). Despite these compounds being
structurally comparable and profiling very similarly in terms
of their rat iv PK values, cyclopropyl compound 47 showed a
significantly greater oral exposure than did compound 46, which
confirms the mouse data. Accordingly, compound 47 was
advanced to further pharmacokinetic analysis in dogs. Table 7
highlights the PK profile of 47 in dog following its dosing iv at
2.5 mg/kg and po at 10 mg/kg in a methylcellulose vehicle as
a suspension formulation. The data show that this compound
retains an excellent level of bioavailability that is similar to that
seen in rat but has a lower relative clearance that equates to
approximately 16% hepatic blood flow (68% in rat). As such,
this pharmacokinetic profile provided therapeutically relevant
levels of PARP inhibitor over at least 4 h in this species.
Cyclopropylamide analog 47 was shown to possess good
exposure together with attributes of potency and physicochem-
istry such that this compound was advanced to further analysis

6586 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Menear et al.
Figure 3. Cell survival (measured as a survival fraction) of breast
cancer cell lines treated with PARP inhibitor 47. The BRCA1- and
BRCA2-proficient lines were Hs578T, MDA-MB-231, and T47D,
whereas the BRCA1-deficient lines were MDA-MB-436 and HCC1937.
Figure 2. (a) Potentiation of methyl methanesulphonate (MMS) cell
killing of cultured SW620 cells in combination with compound 47.
Increasing concentrations of MMS were coincubated with or without
the PARP inhibitor at single concentrations ranging from 1 to 300 nM.
(b) PARP activity in SW620 tumor cell lysates treated with increasing
nM concentrations of compound 47. PARP activity was quantified by
the use of a PAR formation assay. (See the Experimental Section.)
Figure 4. Antitumor efficacy of compound 47 in combination with
temozolomide (TMZ) in an SW620 tumor model. Mice were orally
dosed once daily for 5 consecutive days (days 0-4 are indicated by a
short line), and compound 47 was administered 45 min before TMZ.
Tumor volumes are shown relative to the initial tumor volume prior to
dosing. Error bars represent SEM.
of its cellular potency and in vivo efficacy to identify it as a
potential clinical candidate.
In Vitro Cellular Potency
The cellular potency of 47 was evaluated in a number of in
vitro models. Initial studies identified 47 as an effective agent
to potentiate the cell killing by alkylating agent MMS. Following
the establishment of a growth-inhibition curve for MMS on
SW620 cells, the addition of the PARP-1 inhibitor at nM
concentrations dose dependently increased the effectiveness of
MMS (Figure 2a). The level of potentiation was seen to plateau
around 100 nM concentration, which indicates that the maximal
cellular activity for 47 in combination with MMS lies within
this range. We confirmed this by directly measuring PARP-1
inhibitory activity in cells by using a PAR formation assay in
which 47 was applied to SW620 cell lysates at a similar
concentration range and identified the IC50 for PARP-1 inhibition
to be around 6 nM and the total ablation of PARP-1 activity to
be at concentrations of 30-100 nM (Figure 2b).
was observed for the TMZ plus compound 47 combination
(mean values given as relative tumor volumes (RTV), Figure
4). This equated to over 80% tumor growth inhibition throughout
the entire terminal phase of the study between TMZ treatment
and the combination. The time to reach 2× RTVs for TMZ
and combination treatment was 44 and 70 days, respectively
(59% increase in latency). Higher RTV values such as 4× RTV
(two doublings) could not be comparably assessed because
tumors that were treated with the combination treatment did
not attain this size during the duration of the study, which clearly
demonstrates the pronounced potentiation of TMZ activity by
compound 47 (Figure 4). TMZ was well tolerated with a
maximum mean body weight loss of 6% on day 7 (3 days after
dosing concluded) and full recovery within a week. Likewise,
when administered in combination, the PARP inhibitor did not
exacerbate the systemic toxicity of TMZ, with a maximum mean
body weight loss of 9% on day 6 with full recovery of body
weight within 3 days and with no mortalities (>20% weight
loss), which signifies that the combination therapy was well
tolerated under this dosing regimen.
Consistent with previous data,24 BRCA1-deficient cell lines
MDA-MB-463 and HCC1937 were hypersensitive to PARP
inhibition by compound 47 in comparison with BRCA1- and
BRCA2-proficient cells (Figure 3). Consequently, the clinical
use of compound 47 is being explored in BRCA-deficient
tumors.35
In Vivo Efficacy
Conclusions
On the basis of our in vitro cell data, the ability of compound
47 to potentiate the antitumor activity of the methylating
chemotherapeutic agent, TMZ, was evaluated in an SW620
tumor model.
Animals bearing SW620 xenografted tumors were treated
with compound 47 (10 mg/kg, po) in combination with TMZ
(50 mg/kg, po) once daily for 5 consecutive days, after which
the tumors were left to grow out. A considerable inhibition of
tumor volumes as compared with that of the TMZ alone group
The optimization of a series of substituted 4-benzyl-2H-
phthalazin-1-ones has led to the identification of an array of
highly potent PARP-1 inhibitors that possess cellular potency.
Through a combination of parallel synthesis and subtle side-
chain modification, compounds from both a homopiperazine and
a piperazine series have been elaborated to introduce good oral
bioavailability. Furthermore the introduction of a C-4 fluorine
onto the benzyl linker has been shown to improve PF50 over

NoVel BioaVailable Inhibitor of PARP-1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6587
High-resolution mass spectrometry was carried out on a Thermo
LTQ XL Orbitrap mass spectrometer fitted with an ESI source that
was used for all analyses (ThermoFisher Scientific). The ESI source
was operated in positive-ionization mode with the following source
conditions: a capillary temperature of 275 °C; sheath, auxiliary,
and sweep gas flows of 30, 3, and 5, respectively (arbitrary units);
and needle, capillary, and tube lens voltages of 4000, 48, and 75
V, respectively. We used the mass spectrometer in a data-dependent
mode by using the following scan cycle: (1) scan 1, full scan
100-800 amu; (2) scan 2, MS/MS scan of the most intense ion
detected in scan 1; and (3) scan 3, MS/MS of the most intense ion
detected in scan 2. All data were collected at 30K resolution for
scan 1 (50% valley definition) and at 7.5K resolution for scan modes
2 and 3. Accurate mass measurement was maintained by the use
of three solvent/gas background impurities as lock masses: butyl-
benzenesulfonamide (m/z 214.08963), diisooctyl phthalte (m/z
the corresponding des-fluoro analogs consistently. A medium
throughput murine pharmacokinetic model was exploited to
highlight key compounds for a more extensive analysis, and
this approach lead to the identification of 47. Further preclinical
profiling of 47 in cell lines with genetic lesions in the HR-
repair pathway (BRCA1 and 2), together with in vivo activity
in combination with the DNA-damaging agent TMZ, resulted
in the selection of 47 for clinical development. Compound 47
(KU-0059436, AZD2281) is currently undergoing phase II
clinical trials as a monotherapy in BRCA-deficient breast or
ovarian cancer patients and in combination with selected
cytotoxic agents over a range of tumors.
Experimental Section
All experiments were carried out under an inert atmosphere and
at room temperature, unless otherwise stated. The purities of
compounds for biological testing were assessed by one of two
analytical LC-MS methods. The first method utilized a Spectra
System P4000 pump and a Jones Genesis C18 column (4 µm, 50
× 4.6 mm2). Mobile phases A (0.1% formic acid in water) and B
(acetonitrile) were used in a gradient of 5% B for 1 min, which
rose to 98% B after 5 min, and were held for 3 min at a flow rate
of 2 mL/min. Detection was by a TSP UV 6000LP detector at 254
nm UV and a 210-600 nm range PDA. We used a Finnigan LCQ
mass spectrometer operating in positive-ion electrospray mode. The
second method utilized an Agilent 1100 apparatus, Waters Alliance
HT (2790 and 2795) equipment, or an HP1100 pump and diode
array with a CTC autosampler run on a Phenomenex Gemini C18
column (5 mm, 50 × 2 mm2) eluting with acidic eluant. (For
example, by the use of a gradient, over 4 min, between 0 and 95%
water/acetonitrile with 5% of 1% formic acid in 50:50 water/
acetonitrile (v/v) mixture the MS component comprised a Waters
ZQ mass spectrometer that scanned over an appropriate mass range.)
Chromatograms for electrospray ionization (ESI) positive and
negative base peak intensity and a UV total absorption chromato-
gram from 220-300 nm were generated, and values for m/z are
given; generally, only ions that indicate the parent mass are reported,
and unless otherwise stated, the value quoted is (M + H)+ for
positive-ion mode and (M - H)- for negative-ion mode.
+
391.284286), and a siloxane adduct, [Si(CH3)2O]8NH4 (m/z
610.184156).
3-[(4-Oxo-3H-phthalazin-1-yl)methyl]benzoic Acid (7). To a
stirred suspension of 1 (51.0 g, 0.38 mol) and 2 (52.0 g, 0.39 mol)
in ethyl propionate (200 mL) was added a solution of sodium
methoxide in methanol (25% wt, 1.530 mol) over 40 min, and the
temperature was maintained below 30 °C. The reaction was then
heated under reflux for 1 h before it was diluted with methanol
(100 mL) and further heated for 1 h. The mixture was cooled and
concentrated in vacuo. Water (1 L) was added to the resulting
suspension, which was filtered. The filtrate was washed with diethyl
ether (3 × 200 mL) and was acidified with acetic acid (110 mL),
and the suspension was stirred for 15 min. The product was
collected by filtration, washed with water (100 mL), and dried in
air to produce 69.2 g of 3-(1,3-dioxoinden-2-yl)benzonitrile 3 (94%)
as a red solid (95% purity by HPLC, material used without further
purification). A suspension of 3 (48.3 g. 0.194 mol) in aqueous
NaOH (39.1 g, 0.98 mol) in water (350 mL) was heated to 100 °C
for 72 h. The solution was cooled to 20 °C and was acidified with
HCl (2 N) to pH 2. After 15 min, the solid was filtered and was
washed with water (3 × 300 mL), hexane (2 × 200 mL), and diethyl
ether (2 × 150 mL) and was dried to yield a pale-pink powder.
The powder was suspended in hydrazine hydrate (250 mL, 5.0 mol)
and was heated to 100 °C for 3 h. The reaction was cooled to 20
°C and was filtered. The filtrate was diluted with saturated aqueous
NaHCO3 solution (50 mL), was washed with ethyl acetate (2 ×
100 mL), and was acidified with 6 N HCl (ca. 75 mL) to pH 4.
The resulting pale-yellow suspension was stirred for 10 min, and
the product was collected by filtration and was washed with water
(4 × 150 mL), hexane (3 × 200 mL), diethyl ether (2 × 100 mL),
and acetone (2 × 100 mL) to produce 27.7 g of 7 (55%) as an
off-white powder.
2-Fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]benzoic Acid
(8). Dimethylphosphite (97.3 mL, 1.06 mol) was added dropwise
to a solution of sodium methoxide (463 g, 1.14 mol) in methanol
(1 L) at 0 °C. 2-Carboxybenzaldehyde (135 g, 0.90 mol) was then
added portionwise to the reaction mixture over 20 min, and the
temperature was maintained below 5 °C. The resulting orange
solution was warmed to 20 °C over 2 h. Methanesulphonic acid
(81.6 mL, 1.26 mol) was added to the reaction dropwise over 35
min, and the resulting white suspension was evaporated in vacuo.
The white residue was treated with water (550 mL) and was
extracted with DCM (3 × 400 mL). The combined organic extracts
were backwashed with water (2 × 100 mL), were dried over
MgSO4, and were evaporated in vacuo to produce 194.0 g of
3-dimethoxyphosphoryl-3H-2-benzofuran-1-one 4 (89%) as a crys-
talline white solid. The material was carried forward without further
purification. To a mixture of 4 (35.0 g, 0.14 mol) and 2-fluoro-5-
formylbenzonitrile 5 (20.9 g, 0.14 mol) in THF (330 mL) was added
triethylamine (14.0 mL, 0.14 mol) dropwise over 25 min, and the
temperature was maintained below 15 °C. The reaction mixture
was slowly warmed to 20 °C over 1 h and was then concentrated
in vacuo. The residue was slurried in water (250 mL) for 30 min,
and the solid was collected by filtration, was washed with water (2
× 30 mL), hexane (2 × 30 mL), and diethyl ether (2 × 30 mL),
We conducted purification by preparative HPLC/MS on a Waters
mass-directed purification system by utilizing a Waters 600 LC
pump, a Waters Xterra C18 column (5 µm, 19 × 50 mm2), and a
Micromass ZQ mass spectrometer operating in positive-ion ESI
mode. Mobile phases A (0.1% formic acid in water) and B (0.1%
formic acid in acetonitrile) were used in a gradient (5% B to 100%
over 7 min; held for 3 min at a flow rate of 20 mL/min). The
separation of the drug standards and their metabolites was carried
out by the use of a Gemini C18 column (5 µm, 250 × 2 mm2 i.d.)
(Phenomenex, Macclesfield, U.K.). The column was heated to 50
°C, and the mobile-phase flow rate was 300 µL/min. For all
separations eluant A was 10 mM aqueous ammonium acetate.
Eluant B was 10 mM ammonium acetate in methanol. (All
chemicals and solvents were obtained from Fisher Scientific,
Loughborough, U.K.) Each sample (10 µL) was injected and
subjected to the following mobile phase. We used an HPLC system
that comprised the following parts: an autosampler (CTC Analytics),
an HTS PAL apparatus (Presearch), a Thermo Surveyor Plus MS
pump and photodiode array detector (ThermoFisher Scientific), and
a Thermo Surveyor Plus PDA (ThermoFisher Scientific). The
photodiode array detector was scanned over a wavelength range
of 190-350 nm. All results are shown at 300-305 nm.
1H NMR and 13C NMR were recorded by the use of a Bruker
DPX 300 spectrometer at 300 and 75 MHz, respectively. Chemical
shifts were reported in parts per million (ppm) on the δ scale relative
to tetramethylsilane internal standard, and peak multiplicities are
expressed as follows: s, singlet; d, doublet; dd, doublet of doublets;
t, triplet; br s, broad singlet; and m, multiplet. Unless otherwise
stated, all samples were dissolved in DMSO-d6. NMR and mass
spectra were run on isolated intermediates and final products and
were consistent with the proposed structures.

6588 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Menear et al.
and was dried to produce 37.2 g of 2-fluoro-5-[(Z/E)-(3-oxo-2-
benzofuran-1-ylidene)methyl]benzonitrile 6 (96%) as a 75:25
mixture of E and Z isomers. The material was carried forward
without separation of the isomers. As such, to a stirred suspension
of 6 (37.2 g, 0.14 mol) in water (200 mL) was added aqueous NaOH
(13 N, 50 mL) with subsequent heating to 90 °C for 1 h. The
reaction mixture was partially cooled to 70 °C and hydrazine hydrate
(100 mL, 2.0 mol) added, and the mixture was stirred for 18 h at
70 °C. The reaction was cooled to ambient temperature and was
acidified with HCl (8 N, ca. 80 mL) to pH 4. After 10 min, the
suspension was filtered and was washed with water (2 × 60 mL),
followed by diethyl ether (3 × 50 mL) and was dried to produce
30.1 g of 8 (77%) as a white powder.
tert-Butyl 4-[3-[(4-Oxo-3H-phthalazin-1-yl)methyl]benzoyl]pip-
erazine-1-carboxylate (9). To a solution of 7 (5.0 g, 0.178 mol),
tert-butyl-1-piperazine carboxylate (3.65 g, 0.196 mol), HBTU (6.82
g, 0.19 mol), and DIPEA (6.1 mL, 0.38 mol) were added to DMA
(60 mL), and the mixture was stirred for 1 h. Water (100 mL) was
added to the mixture, which was further stirred 1 h. The suspension
was cooled to room temperature, was filtered, was washed with
ice water (2 × 15 mL) and diethyl ether (2 × 15 mL), and was
dried to produce 7.3 g of 9 (91%) as a white crystalline solid.
tert-Butyl 4-[3-[(4-Oxo-3H-phthalazin-1-yl)methyl]benzoyl]-
1,4-diazepane-1-carboxylate (10). A solution of 7 (0.56 g, 2.0
mmol), tert-butyl-1-homopiperazine carboxylate (0.48 g, 2.4 mmol),
HBTU (0.985 g, 2.60 mmol), and triethylamine (0.61 mL, 4.4
mmol) in DMF (3.5 mL) was stirred at ambient temperature for
18 h. Water (5 mL) was added in one portion, and the mixture was
cooled (5 °C) for 1 h. The suspension was filtered, was washed
with DMF-H2O (1:1) (2 × 2 mL) and ether (2 × 2 mL), and was
dried to produce 891 mg of 10 (97%) as a white solid.
tert-Butyl 4-[2-Fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]-
benzoyl]piperazine-1-carboxylate (11). To 8 (8.9 g, 30.0 mmol),
tert-butyl-1-homopiperazine carboxylate (6.70 g, 36.0 mmol),
HBTU (13.6 g, 36.0 mmol), and triethylamine (8.3 mL, 60.0 mmol)
was added DMF (60 mL), and the mixture was stirred for 2 h.
Water (60 mL) was added, and the mixture was cooled to 5 °C for
>1 h. The suspension was filtered, was washed with ice-cold
DMF-H2O (1:1) (2 × 20 mL), ice-cold water (2 × 20 mL), cold
IPA (2 × 20 mL), and cold diethyl ether (2 × 20 mL), and was
dried to produce 13.6 g of 11 (96%) as a white solid.
tert-Butyl 4-[3-[(4-Oxo-3H-phthalazin-1-yl)methyl]benzoyl]-
1,4-diazepane-1-carboxylate (12). A solution of 8 (1.50 g, 5.0
mmol), tert-butyl-1-homopiperazine carboxylate (1.20 g, 6.0 mmol),
HBTU (2.64 g, 6.5 mmol), and triethylamine (1.5 mL, 11.0 mmol)
in DMF (10 mL) was stirred at room temperature for 2 h. Water
(10 mL) was added, and the mixture was cooled to 5 °C over 1 h.
The suspension was filtered, was washed with ice-cold DMF-H2O
(1:1) (2 × 5 mL), cold water (2 × 5 mL), cold IPA (2 × 5 mL),
and cold ether (2 × 5 mL), and was dried to yield 2.4 g of 12
(97%) as a cream solid.
2H). FTMS + PESI m/z: calcd for C21H22FN4O3, 381.172130;
found, 381.17303.
Method Used to Prepare 15 and 17. 4-[[3-[4-(2-Hydroxy-
ethyl)-1,4-diazepane-1-carbonyl]phenyl]methyl]-2H-phthalazin-
1-one (15). To a solution of 7 (67 mg, 0.24 mmol), DIPEA (0.7
mL, 4.0 mmol), and HBTU (95 mg, 0.25 mmol) in DMA (1 mL)
at ambient temperature was added a solution of 2-[1,4]diazepan-
1-ylethanol (0.26 mmol) in DMA (0.5 mL). The reaction mixture
was stirred at room temperature for 16 h and was then submitted
for preparative HPLC purification to produce 15.
A similar procedure was used to prepare compound 17.
Method Used to Prepare 16 and 21. 4-[[4-Fluoro-3-[4-(2-
hydroxyethyl)-1,4-diazepane-1-carbonyl]phenyl]methyl]-2H-ph-
thalazin-1-one (16). To a solution of 8 (50 mg, 0.171 mmol),
DIPEA (0.6 mL, 3.4 mmol), and HBTU (71 mg, 0.19 mmol) in
DMA (1 mL) at ambient temperature was added an appropriate
solution of 2-[1,4]diazepan-1-ylethanol (0.19 mmol) in DMA (0.5
mL). The reaction mixture was stirred at room temperature for 16 h
and was then submitted for preparative HPLC purification: 1H NMR
(DMSO-d6, δ): 12.58 (s, 1H), 8.33-8.21 (m, 1H), 7.98-7.75 (m,
3H), 7.52-7.16 (m, 3H), 4.68 (d, J ) 6.1 Hz, 1H), 4.33 (s, 2H),
4.06-3.87 (m, 2H), 3.80-3.63 (m, 2H), 3.63-3.31 (m, 2H),
3.00-2.88 (m, 2H), 2.88-2.75 (m, 3H), 2.10-1.90 (m, 1H),
1.82-1.73 (m, 2H). FTMS + PESI m/z: calcd for C23H26FN4O3,
425.19834; found, 425.19755.
A similar procedure was used to prepare compound 21.
4-[[3-(4-Ethylsulfonyl-1,4-diazepane-1-carbonyl)phenyl]methyl]-
2H-phthalazin-1-one (18). To a solution of 10a (72 mg, 0.200
mmol) and triethylamine (41 µL, 0.3 mmol) in dry DCM (1 mL)
was added ethane sulfonyl chloride (29 mg, 0.23 mmol). The
mixture was stirred at ambient temperature for 4 h and was then
quenched by the addition of water (0.5 mL). The crude mixture
was then purified by preparative HPLC purification: 1H NMR
(DMSO-d6, δ): 12.58 (s, 1H), 8.34-8.20 (m, 1H), 8.02-7.75 (m,
3H), 7.49-7.15 (m, 4H), 4.35 (s, 2H), 3.77-3.58 (m, 2H),
3.53-3.21 (m, 5H), 3.16-2.97 (m, 2H), 1.82 (s, 1H), 1.50 (s, 1H),
1.26-1.11 (m, 3H). FTMS + PESI m/z: calcd for C23H27N4O4S,
455.17475; found, 455.17376.
4-[[3-[4-(Morpholine-4-carbonyl)-1,4-diazepane-1-carbon-
yl]phenyl]methyl]-2H-phthalazin-1-one (19). To a solution of 10a
(72 mg, 0.200 mmol) and Et3N (33 µL, 0.24 mmol) in dry DCM
(1 mL) was added 4-morpholinecarbonyl chloride (33 mg, 0.240
mmol). The mixture was stirred at ambient temperature for 10 h
and was quenched by the addition of methanol (0.5 mL). The crude
mixture was then purified by preparative HPLC purification to
produce 19.
4-[[4-Fluoro-3-[4-(morpholine-4-carbonyl)-1,4-diazepane-1-
carbonyl]phenyl]methyl]-2H-phthalazin-1-one (20). To a solution
of 14 (38 mg, 0.100 mmol) and triethylamine (33 µL, 0.24 mmol)
in dry DCM (1 mL) was added 4-morpholinecarbonyl chloride (15
mg, 0.11 mmol). The mixture was stirred at ambient temperature
for 4 h and was then quenched by the addition of methanol (0.5
mL). The crude product was then purified by preparative HPLC:
1H NMR (300 MHz, DMSO-d6, δ): 12.56 (s, 1H), 8.33-8.19 (m,
1H), 8.02-7.77 (m, 3H), 7.46-7.35 (m, 1H), 7.31 (dd, J ) 6.4,
2.2 Hz, 1H), 7.27-7.15 (m, 1H), 4.32 (s, 2H), 3.82-3.40 (m, 8H),
3.37-3.16 (m, 6H), 3.12-2.95 (m, 4H). FTMS + PESI m/z: calcd
for C26H29FN5O4, 494.21980; found, 494.21828.
4-[[3-(1,4-Diazepane-1-carbonyl)-phenyl]methyl]-2H-phthalazin-
1-one (10a). To a cold (5 °C) solution of 10 (0.50 g, 1.08 mmol)
in DCM (10 mL) was added dropwise TFA (2 mL, 10.8 mmol).
After 2 h, the reaction was reduced to dryness in vacuo and was
resuspended in DCM (10 mL). Triethylamine (ca. 0.5 mL) was
added dropwise. The mixture was then absorbed onto silica gel
and was subjected to SiO2 chromatography with EtOAc/MeOH (7:
1) as the eluant to afford 0.33 g of 10a (83%) as a white solid.
4-[[3-(1,4-Diazepane-1-carbonyl)-4-fluorophenyl]methyl]-2H-
phthalazin-1-one (14). HCl (6 N, 10 mL) was added dropwise to
a solution of 12 (2.40 g, 5 mmol) in ethanol (5 mL). The reaction
was stirred for 3 h and was then concentrated in vacuo (ca. 5 mL).
The pH of the mixture was adjusted to 10 by the use of aqueous
NH3OH. The mixture was then extracted with DCM (3 × 20 mL),
and combined extracts were washed with water (1 × 20 mL) and
were dried over Na2SO4. After the mixture stood overnight, 14 was
isolated by filtration (1.25 g, 66%) as a white crystalline solid (99%
purity by HPLC). 1H NMR (DMSO-d6, δ): 12.58 (s, 1H),
8.28-8.19 (m, 1H), 8.04-7.72 (m, 3H), 7.51-7.10 (m, 3H), 4.31
(s, 2H), 3.81-3.54 (m, 2H), 3.39-2.64 (m, 7H), 2.04-1.55 (m,
4-[[3-[4-(Cyclopropanecarbonyl)-1,4-diazepane-1-carbonyl]-
4-fluorophenyl]methyl]-2H-phthalazin-1-one (22). To a solution
of 14 (38 mg, 0.100 mmol) and Et3N (33 µL, 0.24 mmol) in dry
DCM (1 mL) was added cyclopropanecarbonyl chloride (11 mg,
0.11 mmol). The mixture was stirred at ambient temperature for
8 h and was then quenched by addition of methanol (0.5 mL). The
1
crude product was then purified by preparative HPLC: H NMR
(300 MHz, DMSO-d6, δ): 12.56 (s, 1H), 8.26 (d, J ) 7.7 Hz,
1H), 8.04-7.77 (m, 3H), 7.49-7.29 (m, 1H), 7.29-7.14 (m, 2H),
4.32 (s, 2H), 3.83 (m, 1H), 3.73-3.54 (m, 3H), 3.43 (m, 2H), 3.26
(m, 4H), 1.55-1.28 (m, 1H), 0.70 (m, 4H). FTMS + PESI m/z:
calcd for C25H26FN4O3, 449.19834; found, 449.19752.

NoVel BioaVailable Inhibitor of PARP-1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6589
4-[[3-(Piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-
1-one (23). To a cold (5 °C) solution of 9 (5.3 g, 11.9 mmol) in
DCM (60 mL) was added dropwise TFA (8 mL, 108 mmol). After
2 h, the reaction was reduced to dryness in vacuo and was
resuspended in DCM (60 mL). Triethylamine (ca. 3 mL, 21 mmol)
was added dropwise. The mixture was then absorbed onto silica
gel and was subjected to SiO2 chromatography with the eluant
EtOAc/MeOH (7:1) to afford 3.85 g of 23 (92%) as a white solid
(99% purity by HPLC).
4-[[4-Fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-ph-
thalazin-1-one (24). HCl (6 N, 60 mL). was added to a solution
of 11 (13.6 g, 29 mmol) in EtOH (30 mL) at room temperature.
The reaction was stirred for 3 h and was then concentrated in vacuo
(ca. 50 mL). The pH of the mixture was adjusted to 10 by the use
of aqueous ammonium hydroxide (4 N). The mixture was then
extracted with DCM (3 × 50 mL), and combined extracts were
washed with water (50 mL) and were dried over Na2SO4. After
the mixture stood overnight, 24 was isolated by filtration (9.9 g,
92%) as a white crystalline solid (99% purity by HPLC).
General Method Used to Prepare 25, 27, 33, 35, 37, and 39.
4-[[3-(4-Pyridin-2-ylpiperazine-1-carbonyl)phenyl]methyl]-2H-
phthalazin-1-one (25). To a solution of 7 (48 mg, 0.171 mmol) in
DMA (1 mL) were added DIPEA (0.6 mL, 3.4 mmol) and HBTU
(71 mg, 0.188 mmol), followed by an appropriate solution of
pyridine-2-ylamine (0.188 mmol) in DMA (0.5 mL). The reaction
mixture was stirred at room temperature for 16 h and was then
submitted for preparative HPLC purification to produce 25. 1H
NMR (DMSO-d6, δ): 12.58 (s, 1H), 8.27 (dd, J ) 0.9, 7.5 Hz,
1H), 8.13 (dd, J ) 1.2, 4.8 Hz, 1H), 7.98 (m, 1H), 7.86 (m, 2H),
7.56 (m, 1H), 7.40 (m, 3H), 7.27 (m, 1H), 6.82 (d, J ) 8.7 Hz,
1H), 6.67 (dd, J ) 5.1, 1.5 Hz, 1H), 4.37 (s, 2H), 3.31 (m, 8H).
FTMS + PESI m/z: calcd for C25H24N5O2, 426.19245; found,
426.19151.
General Method Used to Prepare 42, 43, and 44. 4-[[4-
Fluoro-3-(4-propylpiperazine-1-carbonyl)phenyl]methyl]-2H-
phthalazin-1-one (42). To a solution of 24 (50 mg, 0.144 mmol)
in dry THF (2 mL) were added DIPEA (25 µL, 0.146 mmol) and
the appropriate 1-bromopropane (0.075 mmol). The reaction mixture
was heated at 50 °C for 16 h and was then submitted for preparative
HPLC purification to produce 42: 1H NMR (DMSO-d6, δ): 12.57
(s, 1H), 8.33-8.20 (m, 1H), 8.00-7.78 (m, 3H), 7.51-7.14 (m,
3H), 4.33 (s, 2H), 3.74-3.52 (m, 2H), 3.19 (s, 2H), 2.63-2.26
(m, 4H) 1.46 (dd, J ) 14.9, 7.4 Hz, 2H), 0.86 (t, J ) 7.4 Hz, 3H).
FTMS + PESI m/z: calcd for C23H26FN4O2, 409.20343; found,
409.20230.
A similar procedure was used to prepare compounds 43 and 44.
General Method Used to Prepare 26, 28, 34, 36, 38, 40, 41,
and 45-51. 4-[[3-[4-(Cyclopropanecarbonyl)piperazine-1-car-
bonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one (47). To a
solution of acid 7 (50 mg, 0.168 mmol) in DMA (1 mL) were added
DIPEA (56 µL, 0.336 mmol) and HBTU (64 mg, 0.170 mmol),
followed by cyclopropylpiperazine-1-ylmethanone (0.170 mmol).
The reaction mixture was stirred at room temperature for 16 h and
was then submitted for preparative HPLC purification to produce
1
47: H NMR (DMSO-d6): 12.57 (s, 1H), 8.27 (dd, J ) 7.7, 1.2
Hz, 1H), 7.97 (dd, J ) 7.7, 1.2 Hz, 1H), 7.94-7.78 (m, 2H),
7.49-7.40 (m, 1H), 7.42-7.34 (m, 1H), 7.24 (t, J ) 9.0 Hz, 1H),
4.34 (s, 2H), 3.86-3.10 (m, 8H), 2.09-1.82 (m, 1H), 0.74 (m,
4H). FTMS + PESI m/z: calcd for C24H24FN4O3, 435.18269; found,
435.18199.
A similar procedure was used to prepare compounds 26, 28, 34,
36, 38, 40, 41, 45, 46, and 48-51.
Solubility Measurements. The thermodynamic solubility of a
research compound is measured under standard conditions. A known
amount of the compound is stirred in 0.1 M phosphate buffer (pH
7.4) at constant temperature (25 °C) for 24 h. The supernatant is
then separated from the undissolved material by centrifugation and
is subsequently analyzed and quantified against a standard of known
concentration in DMSO by the use of generic HPLC-UV
methodology coupled to mass spectral peak identification.
Pharmacokinetics. We carried out pharmacokinetics determina-
tions in mouse, rat, and dog. All doses of the compounds were
given as a single dose either intravenously (iv) or orally (po; see
dose level, as described in Tables 6 and 7). For the iv studies, the
compounds were formulated in a mixture of 10% DMSO/10%
cyclodextrin in PBS. For the oral studies, the compounds were also
predominantly formulated as a solution in 10% DMSO/10%
cyclodextrin in PBS except for compound 47, where a suspension
in methylcellulose/PBS was used for oral dosing in dogs.
In Vitro Isolated Enzyme Assay. This assay determined the
ability of test compounds to inhibit PARP-1 enzyme activity. The
method that was used was as reported.43 We measured PARP-2
activity inhibition by using a variation of the PARP-1 assay in which
PARP-2 protein (recombinant) was bound down by a PARP-2
specific antibody in a 96-well white-walled plate. PARP-2 activity
was measured following 3H-NAD+ DNA additions. After washing,
scintillant was added to measure 3H-incorporated ribosylations. For
tankyrase-1, an AlphaScreen (Perkin-Elmer) assay was developed
in which HIS-tagged recombinant TANK-1 protein was incubated
with biotinylated NAD+ in a 384-well ProxiPlate assay. Alpha
beads were added to bind the HIS and biotin tags to create a
proximity signal, whereas the inhibition of TANK-1 activity was
directly proportional to the loss of this signal. All experiments were
repeated at least three times.
A similar procedure was used to prepare compounds 27, 33, 35,
37, and 39.
4-[[3-(4-Ethylsulfonylpiperazine-1-carbonyl)phenyl]methyl]-
2H-phthalazin-1-one (29). To a solution of 23 (45 mg, 0.072
mmol) in dry DCM (11 mL) were added DIPEA (25 µL, 0.146
mmol) and ethanesulphonyl chloride (7 µL, 0.075 mmol). The
reaction mixture was stirred at room temperature for 16 h and was
then submitted for preparative HPLC purification to produce 29.
4-[[3-(4-Ethylsulfonylpiperazine-1-carbonyl)-4-fluorophenyl-
]methyl]-2H-phthalazin-1-one (30). To a solution of 24 (25 mg,
0.072 mmol) in dry DCM (1 mL) were added DIPEA (25 µL, 0.146
mmol) and ethanesulphonyl chloride (7 µL, 0.075 mmol). The
reaction mixture was stirred at room temperature for 16 h and was
then submitted for preparative HPLC purification. 1H NMR
(DMSO-d6, δ): 12.58 (s, 1H), 8.27 (dd, J ) 7.2, 1.5 Hz, 1H),
7.95 (dd, J ) 7.2, 1.5 Hz, 1H), 7.92-7.79 (m, 2H), 7.49-7.41 (m,
1H), 7.36 (dd, J ) 6.5, 2.2 Hz, 1H), 7.24 (t, J ) 9.6 Hz, 1H), 4.34
(s, 2H), 3.70 (s, 2H), 3.25 (t, J ) 4.5 Hz, 4H), 3.11-3.02 (m, 4H),
1.21 (t, J ) 7.2 Hz, 3H). FTMS + PESI m/z: calcd for
C22H24FN4O4S, 459.14968; found, 459.14877.
4-[[3-[4-(Morpholine-4-carbonyl)piperazine-1-carbonyl]phe-
nyl]methyl]-2H-phthalazin-1-one (31). To a solution of 23 (25
mg, 0.072 mmol) in dry DCM (1 mL) were added DIPEA (25 µL,
0.15 mmol) and morpholine-4-carbonyl chloride (12 mg, 0.075
mmol). The reaction mixture was stirred at room temperature for
16 h and was then submitted for preparative HPLC purification to
produce 31.
4-[[4-Fluoro-3-[4-(morpholine-4-carbonyl)piperazine-1-car-
bonyl]phenyl]methyl]-2H-phthalazin-1-one (32). To a solution
of 24 (25 mg, 0.072 mmol) in dry DCM (1 mL) were added DIPEA
(25 µL, 0.146 mmol) and morpholine-4-carbonyl chloride (12 mg,
0.075 mmol). The reaction mixture was stirred at room temperature
for 8 h and was then submitted for preparative HPLC purification.
1H NMR (DMSO-d6, δ): 12.58 (s, 1H), 8.34-8.20 (m, H),
8.03-7.75 (m, 3H), 7.49-7.15 (m, 6H), 7.00-6.74 (m, 3H), 4.37
(s, 2H), 3.79-3.10 (m, 8H). FTMS + PESI m/z: calcd for
C25H27FN5O4, 480.20415; found, 480.20294.
In Vitro Cell PF50 Assay. The PF50 value is the potentiation
factor, which is calculated as the ratio of the IC50 of the control
growth with alkylating agent methylmethane sulfonate (MMS)
divided by the IC50 of the MMS combined with the PARP inhibitor.
HeLa B cells were used, and the test compounds were tested at a
fixed 200 nM concentration for screening with MMS. For the testing
of compound 47 on the SW620 colorectal cell line (Figure 2), the
concentrations that were used were 1, 3, 10, 100 and 300 nM. Cell
growth was assessed by the use of the sulforhodamine B (SRB)
assay.44

6590 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Menear et al.
Ex Vivo PARP Activity Assay. SW620 whole-cell protein
extracts were prepared by incubation in extraction buffer (1× PBS,
1% NP-40, protease inhibitor cocktail (Roche), 200 µM DTT) for
10 min at 4 °C. PARP activity was determined by the quantification
of the amount of PAR formation after ex vivo activation. PARP
activation reactions were performed by the use of 65 ng of SW620
whole-cell extract in a reaction mix (50 mM Tris pH 8, 4 mM
MgCl2, 200 µM DTT, 200 µM NAD+, 20 ng/µL DNA) at 30 °C
for 5 min. The amount of PAR that formed in each reaction was
then quantified by the use of the Meso Scale Discovery assay
platform. Data were calculated from triplicate experiments as the
mean percentage of PARP activity relative to vehicle controls (SE
and IC50, which were calculated by the use of XL-FIT 4 software.
Cell Lines and Culture. SW620 colon, A2780 ovarian, HCC1937,
Hs578T, MDA-MB-231, MDA-MB-436, and T47D breast cancer
cell lines were obtained from either ATCC or ECACC repositories.
All cell lines were grown as monolayers in RPMI1640 medium
that was supplemented with 10% v/v FBS, 100 µg/mL penicillin,
and 100 µg/mL streptomycin.
Cell Line Cytotoxicity Assays. The effect of KU-0059436 on
the cell survival of breast cancer cell lines was determined by the
use of clonogenic assays, as previously described.45 Briefly, cells
were seeded in six-well plates and were left to attach overnight.
Vehicle control (DMSO) or increasing concentrations of KU-
0059436 (up to 4 µM) were added to the cells, and the mixture
was left for 7-14 days, depending on the cell type, before surviving
colonies were counted. Data were calculated from triplicate wells
as the mean percentage of cell survival relative to vehicle controls
(SE and IC50, which were calculated by the use of XL-FIT 4
software.
comparisons). From the Jonckheere-Terpstra trend test, we con-
cluded that at day 45 there was a statistically significant effect of
increasing the dose of 47 (10 mg/kg, data shown only in Figure 3)
when used in combination with 50 mg/kg TMZ as compared with
that of TMZ alone (p < 0.0001). This was confirmed by the use of
Wilcoxon rank-sum tests, which compared the combination treat-
ment groups versus TMZ alone to demonstrate statistically signifi-
cant differences between TMZ monotherapy and TMZ in combi-
nation with 47 at 10 mg/kg (p ) 0.007) at day 45.
Acknowledgment. We acknowledge the invaluable technical
assistance of the following: Adrian Moore, Penny Wright, James
Pullen, Barry Pope, David Rudge, Nicholas Barron, Craig
Roberts, Jon Eden, Carlos Fernandez Lence, Joanne Gibney,
Kristel Blackburn, Alexandra Fundo, and Rachael Tuppin. Also,
we thank Madeleine Vickers (Structure Purity Group, Astra-
Zeneca) for all high-resolution mass spectroscopy determination,
Elizabeth Mills (Statistical Sciences Group, Discovery, Astra-
Zeneca) for statistical analysis conducted on the in vivo
experimentation, and Nicola Griffin for help with the manuscript.
Supporting Information Available: High-resolution mass spec-
tra and HPLC purity data for all target compounds and additional
spectroscopic and purity data for intermediates 4, 6-9, 10, 10a,
11, and 12 and target compounds 15, 17, 19, 21, 23, 24, 26-29,
31, 33-41, 43-46, and 48-51. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
Potentiation of MMS Cytotoxicity by 47 Determined by
the Use of Sulforhodamine B Cell Growth Assays. SW620 cells
were seeded in 96-well plates and were left to attach overnight.
Cells were preincubated with vehicle control (DMSO) or with a
single concentration of KU-0059436 (1, 3, 10, 30, 100 or 300 nM)
for 1 h before the addition of increasing concentrations of MMS.
Cells were incubated in the presence of each drug combination for
4 days before cell growth was quantified by the use of an SRB
assay.44 Data were calculated from triplicate wells as the mean
percentage of cell growth relative to KU-0059436-only wells, and
(SE and IC50 were calculated by the use of XL-FIT 4 software.
SW620 cells showed <24% growth inhibition (>76% cell growth)
when only KU-0059436 was used at concentrations below 300 nM
(data not shown).
Mouse Xenografts. Tagged mice were inoculated sc with 5 ×
106 cells in 0.1 mL of PBS to the right flank. Tumors were measured
thrice weekly, and tumor volumes were estimated from the formula
[length/2] × [width2]. For xenograft chemopotentiation studies,
established tumors in each animal were individually normalized to
their size at the start of the experiment, and the data were calculated
as the change in tumor volume relative to the day 0 volume by the
use of the relative tumor volume (RTV) formula, RTV ) TVx/TV0,
where TVx is the tumor volume on any day and TV0 is the tumor
volume at the initiation of dosing (i.e., day 0). In the tumor growth
curves, the mean represents a full complement of animals in the
treatment groups; below this threshold, we ceased plotting.
Dosing Regimen. When the mean tumor volume reached 100
mm3, tumor-bearing mice were randomized into treatment groups
with eight animals in each group being dosed orally once daily for
5 consecutive days (po, q.d., ×5); for the combination therapies,
the PARP inhibitor was administered 45 min before TMZ.
Compound 47 was formulated in solution, and TMZ was formulated
as a homogeneous suspension in corn oil (Sigma); both dosing
solutions were freshly made each day. Mice in the no-treatment
group received both vehicles on a mg/kg basis. Mice were weighed
daily during the dosing phase to calculate the day’s dose and any
signs of body weight loss accurately (20% weight loss led to the
animal being euthanized). Statistical analyses were calculated on
the data only when a full complement of animals was present in
the treatment groups (i.e., day 13 for the vehicle and 47 mono-
therapy groups and day 45 for the TMZ and combination
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