Synthesis and biological evaluation of substituted 4-(thiophen-2-ylmethyl)- 2H-phthalazin-1-ones as potent PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.07.001

We have developed a series of substituted 4-(thiophen-2-ylmethyl)-2H- phthalazin-1-ones as potent PARP-1 inhibitors. Preliminary biological evaluation indicated that most compounds possessed inhibitory potencies comparable to, or higher than AZD-2281. Among these compounds, 18q appeared to be the most notable one, which displayed an 8-fold improvement in enzymatic activity compared to AZD-2281. These efforts lay the foundation for our further investigation.

Full text of DOI:10.1016/j.bmcl.2014.07.001

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3739–3743
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of substituted
4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones as potent PARP-1
inhibitors
Ling-xiao Wang a, , Xin-bo Zhou a, , Meng-liang Xiao a, Ning Jiang b, Feng Liu b, Wen-xia Zhou b,
Xiao-kui Wang a, , Zhi-bing Zheng a, , Song Li a
⇑
⇑
a Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China
b Department of Traditional Chinese Materia Medica and Neuroimmunopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a series of substituted 4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones as potent
PARP-1 inhibitors. Preliminary biological evaluation indicated that most compounds possessed inhibitory
potencies comparable to, or higher than AZD-2281. Among these compounds, 18q appeared to be the
most notable one, which displayed an 8-fold improvement in enzymatic activity compared to AZD-
2281. These efforts lay the foundation for our further investigation.
Received 22 April 2014
Revised 25 June 2014
Accepted 1 July 2014
Available online 5 July 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
PARP-1 inhibitor
4-(Thiophen-2-ylmethyl)-2H-phthalazin-1-
ones
Synthesis
Biological evaluation
Antitumor
Poly(ADP-ribose)polymerase-1 (PARP-1) is an 113 kDa eukary-
otic nuclear enzyme with an important role in the process of
DNA repair.1 When activated by DNA damage, PARP-1 catalyzes
the transfer of ADP-ribose units to nuclear target proteins to facil-
itate DNA repair, using nicotinamide adenine dinucleotide (NAD+)
as substrate.2 Given the biological role of the enzyme, PARP-1
inhibitors have the potential to prevent the repair of DNA damages
that were implicated by radiation and chemotherapeutic agents,
and combination of them with radio- and chemo-therapy to max-
imize the cancer therapeutic benefits has become an attractive
strategy.3 The potential was further highlighted by reports that
BRCA1/2 deficient cancer cells are highly sensitive to PARP-1 inhib-
itors resulting in cell death.4
Over the past three decades, a variety of PARP-1 inhibitors have
been designed and synthesized based on the nicotinamide (1,
Fig. 1) moiety of NAD+ to mimic the substrate–protein interactions
of NAD+ with the enzyme.5,6 These compounds, which usually con-
tain a primary amide or lactam functionality, belong to different
chemical classes such as benzimidazole carboxamides (2), tricyclic
indoles (3), and phthalazinones (4),7 and several of them are in
advanced clinical trials, including ABT-888 (5), AG-14699 (6),
AZD-2281 (Olaparib, 7), etc. However, despite the high interest
in PARP-1 inhibitors, development of a number of them has
stalled,8 and to date, there are no drugs that modulate the target
have reached the market, thus the effort of pursuing novel safe
and effective PARP-1 inhibitors is still needed.
Among above compounds under active study, AZD-2281 is a
representative of phthalazinones. In its structure, phthalazinone
itself demonstrates weak PARP-1 inhibitory activity,9 and benzyl
phthalazinone (8) substituted in the 4-position is the core scaffold
and identified as a moderately potent PARP-1 inhibitor.10 Besides,
substituted piperazines at the meta position of benzyl are condu-
cive to enhancing activity and maintaining good oral bioavailabil-
ity,11 and the substitutions at the para position of benzyl
contribute to block possible metabolism and extend half life.12
On the basis of the above structure–activity relationship of AZD-
2281, we tried to take thienyl instead of phenyl to seek new possi-
bilities and explore alternative chemical templates. Furthermore,
we made an attempt to enhance potency by doing some elabora-
tions at the distal nitrogen of piperazine. Herein, we report a series
of substituted 4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones as
novel PARP-1 inhibitors.
⇑
Corresponding authors. Tel.: +86 10 66931642.
E-mail addresses: wxkcaptain@aliyun.com (X. Wang), zhengzb@bmi.ac.cn
(Z. Zheng).
The synthesis of the target compounds 18a–18u outlined in
Scheme
1 was achieved via amide bond coupling of the
corresponding acid 14 with substituted piperazines 17. The key
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.07.001
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3740
L. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3739–3743
H
N
O
H2N
O
F
N
H3PO4
N
N
H
N
H
N
H
5 ABT-888
6 AG-14699
O
O
N
O
NH2
N
H
H
n
N
H
R
N
R
N
H
HN
HN
R
O
2 Benzimidazole
carboxamides
3 Tricyclic indoles
N
NH2
O
O
NH
N
1 Nicotinamide
NH
N
O
O
F
N
N
O
N
N
S
N
O
R
N
4 Phthalazinone
8 R=Benzyl
R
Compound 18a-u
7 AZD-2281
O
Figure 1. Classes and representatives of PARP-1 inhibitors.
H
11; X=Br
O
b
O
12; X=CN
O
O
O
NH
N
S
a
d
X
c
H
O
O
OH
O
O
O
S
P
O
9
O
S
OH
CN
10
13
14
O
NH
N
O
O
O
O
R
f
e
14
g
O
O
O
N
N
HN
N
N
NH
R
S
N
16a-16u
17a-17u
18a-18u
15
N
R
O
Scheme 1. Synthesis route for target compounds. Reactions and conditions: (a) Dimethyl phosphite, CH3ONa/MeOH, 0 °C, 30–60 min, methanesulfonic acid, 88.4%; (b) CuCN/
CuI, DMF, reflux, 3–4 h, 66.3%; (c) Et3N, anhydrous THF, 20 °C, 16 h, 95.6%; (d) (i) H2O, nitrogen atmosphere, NaOH (aq), 90 °C, 30 min, (ii) NH2NH2ÁH2O, 70 °C, 18 h, 2 N HCl,
94.0%; (e) HOBt/EDCI, DIPEA, DCM, 18 h, RT, 90.5%; (f) TFA/DCM, below 25 °C, 64%; (g) (i) HBTU/DIPEA, DMA, below 25 °C, 48 h, (ii) water, reflux, 1 h, 26.2–76.9%.
intermediate 14 was prepared from 2-formylbenzoic acid 9,
treating 9 with dimethylphosphite furnished intermediate 10.13
Compound 10 was then submitted to a nucleophilic substitution
in the presence of triethylamine with another substrate 12, which
was afforded by treating 11 with CuCN and CuI,14 to give interme-
diate 13. Then the hydrolysis of 13 was occurred under aqueous
NaOH condition, followed by a reaction with hydrazine hydrate
to give the desired carboxylic acid scaffold 14.15 As for the pipera-
zine moiety, N-boc piperazine 15 was coupled to various acids
with HOBT, EDCI and DIPEA to provide reliably high yields of
16a–16u,16 followed by a boc-deprotection in the presence of
trifluoroacetic acid to give secondary amines 17a–17u.17 Finally,
acid 14 was coupled to various substituted piperazines 17 under
standard HBTU/DIPEA conditions to afford the target compounds
18a–18u.18
All target compounds were evaluated in vitro for their PARP-1
enzyme inhibition activity, and ten of them were selected to deter-
mine IC50 values. The results were listed in Table 1.
To begin with, straightforward replacement of the phenyl in
AZD-2281 (IC50: 84.96 nM) with thienyl gave compound 18a
(104.90 nM), and what made us exciting was that 18a exhibited
the same order of magnitude in potency as AZD-2281. Further

L. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3739–3743
3741
Table 1
The structures and PARP-1 enzyme inhibition activity of compounds 18a–18u
O
NH
N
O
S
N
N
R
O
Compd
R
Inhibitiona (%)
89.97
IC50 (nM)
84.96
Compd
R
Inhibitiona (%)
82.57
IC50 (nM)
F
AZD-2281
—
18k
NDb
ND
Cl
Br
18a
18b
73.86
87.06
104.90
ND
18l
81.02
18m
100.11
72.34
O
O
18c
18d
18e
18f
72.39
82.83
81.74
89.02
123.60
78.62
ND
18n
18o
18p
18q
84.51
71.31
80.88
99.13
ND
ND
O
S
O
39.61
10.84
O
94.88
S
18g
18h
98.72
95.42
ND
ND
18r
18s
82.01
66.17
ND
ND
O
NO2
CN
N
18i
18j
66.01
85.93
ND
18t
68.90
75.60
107.80
46.12
N
85.53
18u
a
PARP-1 inhibitory ratio at 1
ND: not determined.
lmol.
b

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L. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3739–3743
increase, respectively, while their 3 and 4-position counterparts
appeared to be less potent PARP-1 inhibitors.
The above discussions have been validated by a docking study
of the most potent compound 18q into the catalytic domain of
human PARP-1 (PDB code: 2RD6), and the result is shown in
Figure 2. The key interactions in the PARP-1 active site, consistent
with previous literature reports,20,21 are highlighted. The main
phthalazinone ring forms strong pi–pi interactions with Tyr-246,
His-201 and Lys-242, and the carboxamide group on it appears
to be involved in key hydrogen-bond interactions with Ser-243
and Gly-202. Figure 3 shows the predicted binding model of
AZD-2281 overlaid with compound 18q, these typical interactions
are also observed in the binding model of AZD-2281 with the
PARP-1 active site. In addition, the thienyl substituent at the distal
position of 18q lies in the deep cavity forming a characteristic pi–pi
interaction with Arg 217, as we hoped initially.
In summary, we have designed and synthesized a novel series of
substituted 4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones as
potent PARP-1 inhibitors. A wide variety of substituents can be tol-
erated, and most compounds possessed inhibitory potencies com-
parable to, or higher than AZD-2281. Among them, 18p, 18q and
18u appeared to be the most potent ones, especially 18q, which
displayed an 8-fold increase in enzymatic activity compared to
AZD-2281. This indicated that our strategy of replacing fluoro-
substituted phenyl in AZD-2281 with thienyl was beneficial. Fur-
ther biological evaluation of these compounds is ongoing and
results will be reported in due course.
Figure 2. Docking of compound 18q in the catalytic domain of human PARP-1 (PDB
code: 2RD6).
modification of the cyclopropylcarbonyl with various aliphatic car-
bonyls as in 18b, 18c and 18d obtained similar results. Thereafter,
we introduced phenyl and 4-methoxy phenyl to 18c to acquire 18e
and 18f, which still showed good activity comparable to AZD-2281.
Prompted by the encouraging results of aliphatic derivatives,
we made some further exploration to introduce various aromatic
carbonyls to piperazine. Firstly, modifications of R group with
substituted phenyls provided compounds 18g–18p. These com-
pounds basically demonstrated maintained or improved potencies,
and only a moderate drop was observed for 18i that contained a
strong electron-withdrawing group nitryl. Among them, 18p
(39.61 nM) displayed 2-fold improvement in potency compared
with AZD-2281. Then we turned our attention to heteroaromatic
derivatives 18q–18u. Except for 2-furyl compound 18s, hetero-
atom existed in the 2-position of the heterocycle resulted in an
enhancement of PARP-1 inhibitory activity compared with AZD-
2281. For instance, the 2-thienyl compound 18q (10.84 nM)19
and 2-pyridyl compound 18o (46.12 nM) exhibited 8 and 2-fold
Acknowledgments
This work was financially supported by the Major Program of
Ministry of Science and Technology of China (No. 2013ZX09J
13103) and the National Natural Science Foundation of China
(No. 81102309).
References and notes
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15. Procedure for the synthesis of the target compound 14: (a) To a mixture of 10
(0.06 mol) and 12 (0.055 mol) in anhydrous THF (130 mL) was added
triethylamine (130 mL) dropwise with temperature maintained below 15 °C.
The mixture was stirred for 17 h at 20 °C and concentrated in vacuo. The
residue was slurried in water (90 mL) and yellow solid was collected by
filtration, was washed with water, hexane, and diethyl ether, and was dried to
produce 13 in 95.6% yield as a 50:50 mixture of E and Z isomers. The material
Figure 3. Predicted binding model of AZD-2281 overlaid with compound 18q in the
active site of human PARP-1 (PDB code: 2RD6). Key amino acids are depicted as
stick model with the atoms colored as carbon-gray, hydrogen-white, nitrogen-
purple and oxygen-red. Ligands are also represented as stick and colored by atoms
with AZD-2281 colored as carbon-deep blue and compound 18q colored as carbon-
baby blue.
was carried forward without separation of the isomers. (b) To
a stirred
suspension of 13 (0.052 mol) in water (60 mL) was added aqueous NaOH

L. Wang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3739–3743
3743
(12.5 mol/L) under nitrogen atmosphere, with subsequent heating to 90 °C for
30 min. The reaction mixture was then cooled to 70 °C and hydrazine hydrate
(50.2 mL) added, and the mixture was stirred for 18 h at 70 °C with nitrogen
atmosphere removed. The reaction was cooled to ambient temperature and
was acidified with HCl (2 mol/L) to pH 4. After 30 min stirred, the suspension
was filtered and dried to produce 14 in 94% yield as a light red solid.
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was stirred for 48 h, with the temperature maintained below 25 °C. Then water
(11 mL) added, and the mixture was stirred at reflux for 1 h. After cooled, the
suspension was filtered and then purified by column chromatography to give
target compounds 18a–u in 26.2–76.9% yield.
19. Analytical data for selected final compound 18q: White solid. Mp 198–199 °C. MS
m/z 465.5 (M+H)+, 1H NMR (400 MHz, DMSO-d6) d (ppm) 12.65 (s, 1H), 8.28–
8.26 (d, J = 8, 1H), 8.07–8.05 (d, J = 8, 1H), 7.94–7.90 (t, 1H), 7.86–7.82 (t, 1H),
7.79–7.77 (m, 1H), 7.64 (d, J = 1.2, 1H), 7,44 (d, J = 3.2, 1H), 7.15–7.13 (m, 2H),
4.52 (s, 2H), 3.67–3.59 (m, 8H).
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18. General procedure for the synthesis of the target compounds 18a–18u: To a
solution of 14 (1.8 mmol), 17a–17u (1.9 mmol), HBTU (1.8 mmol), and DIPEA
(1.8 mmol) were added to N,N-Dimethylacetamide (2.2 mL), and the mixture