Rational approaches to discovery of orally active and brain-penetrable quinazolinone inhibitors of poly(ADP-ribose)polymerase

Article abstract of DOI:10.1021/jm0499256

A novel class of quinazolinone derivatives as potent poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors has been discovered. Key to success was application of a rational discovery strategy involving structure-based design, combinatorial chemistry, and classical SAR for improvement of potency and bioavailability. The new inhibitors were shown to bind to the nicotinamide-ribose binding site (NI site) and the adenosine-ribose binding site (AD site) of NAD⁺.

Full text of DOI:10.1021/jm0499256

J . Med. Chem. 2004, 47, 4151-4154
4151
Ta ble 1. Seed and Lead Profiles
Ra tion a l Ap p r oa ch es to Discover y of
Or a lly Active a n d Br a in -P en etr a ble
Qu in a zolin on e In h ibitor s of
P oly(ADP -r ibose)p olym er a se
Kouji Hattori,*,† Yoshiyuki Kido,†
1
2
3
Hirofumi Yamamoto,† J unya Ishida,†
Kazunori Kamijo,† Kenji Murano,† Mitsuru Ohkubo,‡
Takayoshi Kinoshita,‡ Akinori Iwashita,§
Kayoko Mihara,§ Syunji Yamazaki,§
Nobuya Matsuoka,§ Yoshinori Teramura,# and
Hiroshi Miyake†
PARP-1 Inhibitiona
60 ( 0.12 1200 ( 60
Brain/Plasma Concentration in Miceb
IC50 (nM)
65 ( 10
brain (µg/g tissue)
plasma (µg/mL)
ratio (B/P)
0.60 ( 0.035
0.70 ( 0.16
0.86
5.5 ( 2.1
6.7 ( 3.3
0.82
7.0 ( 1.8
1.2 ( 0.15
5.8
Medicinal Chemistry Research Laboratories, Exploratory
Research Laboratories, Medicinal Biology Research
Laboratories, and Biopharmaceutical and Pharmacokinetic
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd.,
2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, J apan
a
The results were presented as the mean ( SE of three
independent experiments. For details of assay, see Supporting
Information. The results were presented as the mean ( SE. The
concentration was measured at 0.5 h by HPLC after ip adminis-
b
tration in mice at 32 mg/kg (n ) 2).
Received J anuary 27, 2004
as shown in Table 1. Quinazolinedione 1 linked with
large substitution exhibits strong potency against PARP-1
with an IC50 of 60 nM; however, it showed poor plasma
concentration after intraperitoneal administration in
mice. While quinazolinone 2 exhibits an appropriately
10-fold higher plasma and brain concentration, it showed
poor potency against PARP-1 (IC50 ) 1200 nM).5 We
attempted to combine the desired traits of quinazolinone
2 with the potency of 1 by addition of a linked tetrahy-
dropyridine moiety. Figure 1 conceptualizes our design
proposal to find a desired lead compound based on
molecular modeling of the human active site of PARP-
1.6 Information from the previously solved X-ray struc-
ture of NAD+ bound to the active site of PARP-1 (Figure
1A)7 led us to rationally create a new inhibitor. It is
strongly suggested that quinazolinedione 1 and quinazoli-
none 2 tightly bind to the nicotinamide-ribose binding
site (NI site) as a critical binding mode for a common
structure motif8 and the 4-phenyl-tetrahydropyridine
moiety of 1 provides secondary contacts to the adenine-
ribose binding site (AD site), resulting in potent IC50
(Figure 1B). We designed the related quinazolinone
analogue 3, which is linked by a similar moiety extended
with a three-carbon unit, by modeling to allow a
maximum fix in the AD site (Figure 1C). Most of the
published inhibitors bind to the NI site and/or the
acceptor site but do not bind to the AD site.9 1 and 3
with a unique side chain are the first inhibitors utilizing
NI and AD binding sites. Lead 3 shows strong potency
with an IC50 of 65 nM, good bioavailability, and a
significantly high brain/plasma concentration ratio (B/P
) 6). To validate our modeling results and to improve
efficiently in the new design, we performed X-ray
crystallography of the catalytic domain of human PARP
complexes with 1, as shown in Figure 2.10 As expected,
inhibitor 1 binds to the NI and the AD subsites of the
donor site. The quinazolidinone core binds to the NI site
by three hydrogen bonds and by a sandwiched hydro-
phobic interaction with the phenyl rings of Tyr907 and
Tyr896. The nitrogen atom of the terahydropyridine
moiety directly binds to CO2H of Asp766 located 3.6 Å
from the nitrogen. The terminal phenyl ring lies in a
deep pocket by van der Waals interaction. This tetrahy-
Abstr a ct: A novel class of quinazolinone derivatives as potent
poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors has been
discovered. Key to success was application of a rational
discovery strategy involving structure-based design, combi-
natorial chemistry, and classical SAR for improvement of
potency and bioavailability. The new inhibitors were shown
to bind to the nicotinamide-ribose binding site (NI site) and
the adenosine-ribose binding site (AD site) of NAD+.
Poly(ADP-ribose)polymerase-1 (PARP-1, EC 2.4.2.30)
is a chromatin-bound nuclear enzyme involved in a
variety of physiological functions related to genomic
repair, including DNA replication and repair, cellular
proliferation and differentiation, and apoptosis.1 PARP-1
functions as a DNA damage sensor and signaling
molecule. Upon binding to DNA breaks, activated PARP
cleaves NAD+ into nicotinamide and ADP-ribose and
polymerizes the latter into nuclear acceptor proteins
including histones, transcription factors, and PARP
itself. A cellular suicide mechanism of necrosis and
apoptosis by PARP activation has been implicated in
the pathogenesis of brain injury and neurodegenerative
disorders, and PARP inhibitors have been shown to be
effective in animal models of stroke, traumatic brain
injury, and Parkinson’s disease.2,3 Therefore, inhibition
of PARP by pharmacological agents may be useful for
the therapy of neurodegenerative disorders and several
other diseases involved in PARP activation. Recently,
several potent PARP inhibitors have been described
using structure-based discovery; however, there remain
problems in terms of the pharmacokinetics and physical
properties.4 In this paper, we describe rational ap-
proaches to the discovery of orally active PARP inhibi-
tors with high brain penetration.
The discovery process was initiated with a high-
throughput random screening of the Fujisawa sample
collection which identified 1 and 2 as seed compounds,
* To whom correspondence should be addressed. Phone: 81-6-6390-
1220. Fax: 81-6-6304-5435. E-mail address: kouji_hattori@
po.fujisawa.co.jp.
† Medicinal Chemistry Research Laboratories.
‡ Exploratory Research Laboratories.
§ Medicinal Biology Research Laboratories.
#
Biopharmaceutical and Pharmacokinetic Research Laboratories.
10.1021/jm0499256 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/16/2004

4152 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Letters
F igu r e 1. Molecular model of NAD+, 1, and 3 in the human PARP-1 catalytic site. Accessible surface of the carbon atoms at the
active sites are drawn as mesh.
Ta ble 2. SAR of Substituted Quinazolidone Analogues
PARP-1
brain (µg/g tissue)b plasma (µg/mL)b
compd
R
IC50 (nM)a
0.5 h, 2.0 h
0.5 h, 2.0 h
4
3
5
6
7
8
9
H
5-Cl
5-F
6-Cl
7-Cl
8-Cl
6,8-diCl
8-Me
8-Et
16
65
16
27
250
26
39
14
66
26
1.90, 0.37
4.60, 2.82
1.62, 0.22
18.6, 14.2
6.12, 2.66
7.99, 8.43
NT
2.54, 0.73
NT
1.15, 0.00
NT
0.68, 0.15
4.18, 2.17
0.37, 0.14
16.2, 15.5
0.08, 0.51
1.28, 1.20
NT
0.94, 0.40
NT
0.24, 0.00
NT
F igu r e 2. Compound 1 bound to the human PARP-1 active
site. 1 is yellow with heteroatoms being blue for nitrogen, red
for oxygen, and light-blue for fluoride. Important residues are
shown stick style.
10
11
12
3-ABc
8-OMe
11200
Sch em e 1. Combinatorial Chemistry for Quinazolidone
Library
a
Values are the averages from at least two independent dose-
response curves, and standard error for PARP inhibition assay is
typically (5% of the mean or less; see Supporting Information.
b
The concentration of inhibitors in brain and plasma determined
in mice following po administration (32 mg/kg, n ) 2) by ex vivo
assay; see Supporting Information. c 3-Aminobenzamide as
standard PARP inhibitor.
a
Ta ble 3. SAR of Pyridine Analogue
dropyridine moiety induces a conformational change at
the bottom of the AD site by motion of Arg878.11
Our initial procedure to prepare quinazolinone ana-
logues linked via appropriate substitution to establish
SARs is outlined in Scheme 1. Combinatorial chemistry
using solid-phase synthesis with Rink amide resin
produced rapidly and efficiently the desired quinazoli-
none derivatives on a 10 mg scale in a total yield of 40-
80%.12,13
We followed basic SAR trends for three segments of
the lead compound to improve the potency and bioavail-
ability with brain penetration in the lead optimization
process. Measurement of PARP inhibitory activity in
vitro was carried out by a standard method using
human recombinant PARP-1.14 Outlined in Table 2 are
the results of several substituted derivatives of the
quinazolinone core. Unsubstituted derivative 4 showed
4 times more potency than lead 3. Improvement in
activity also occurred by addition of substitution at the
6- or 8-position of the quinazolinone, while the addition
of the chloro group at the 7-position (7) was 3 times less
active than 3. In general, addition of a chloro group to
the phenyl ring improved brain concentration over 4.
The activity of 4-phenyltetrahydropyridine derivatives
is outlined in Table 3. 4-Phenylpiperidine 13 and
N-phenylpiperazine 14 had slightly less activity than
4, while transformation from nitrogen into carbon 15
led to a complete loss of potency, suggesting that
nitrogen is a key factor for binding PARP-1. The three-
ring derivative 16 maintained activity, while indole
derivative 17 exhibited a 100-fold higher concentration
in plasma than brain. Elimination of the phenyl ring
18 showed less potency relative to the original com-
pound. The inhibitory activity of phenyl-substituted

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4153
Ta ble 4. SAR of Substituted Benzene Analogues
PARP-1 brain (µg/g tissue) plasma (µg/mL)
compd
R
IC50 (nM)
0.5 h, 2.0 h
0.5 h, 2.0 h
4
19
20
21
22
23
24
25
26
27
28
29
30
31
H
p-F
m-F
o-F
16
8.9
23
37
23
1.90, 0.37
3.54, 1.16
NT
0.68, 0.15
6.83, 3.48
NT
NT
NT
p-Cl
23.9, 9.41
5.31, 2.34
NT
5.61, 3.75
NT
12.1, 2.78
5.40, 0.84
NT
6.05, 1.67
NT
p-Me
p-OH
p-OMe
m-OMe
o-OMe
p-CF3
p-CN
p-Ph
17
12
F igu r e 3. H2O2 induced cytoprotective effects of 32 in PC12
cells (n ) 6): (//) P < 0.01.
8.3
1000
170
25
6.0
34
NT
NT
23.4, 19.5
4.50, 0.54
NT
6.80, 3.27
20.0, 5.20
NT
4-Py
12
2.76, 0.30
14.1, 0.62
Ta ble 5. Pharmacokinetics Profiles of 32
F igu r e 4. MPTP induced parkinson model of 32 in C57BL/6
mouse (n ) 7): 100% of normal and 0% of control; (//) P <0.01;
(/) P < 0.05
po (fasted), n ) 3
iv,a n ) 3
CLtot
in mice. Brain penetration of 32 was good with a B/P
ratio of approximately 5.15 In addition, many recent
PARP inhibitors lack good solubility in water, making
them difficult to utilize in vivo. 32 and the HCl salt
showed good solubility in water (260 and 5600 µg/mL,
respectively).
dose
Cmax ( SE AUC ( SE t1/2
(µg/mL) (µg‚h/mL) (h) (mL/min/kg) (%)
â
BAb
(mg/kg)
mouse
rat
dog
10
10
1.0
0.30 ( 0.14 NT
1.33 ( 0.26 2.52 ( 0.13 1.4
0.28 ( 0.07 3.16 ( 1.30 6.0
NT
NT
13.5
4.8
NT
20
70
Brain/Plasma Concentration in Mice,c n ) 2
Next, we examined the in vitro and in vivo neuro-
protective action of 32. We have already confirmed that
exposure to H2O2 cytotoxicity induces PARP activation
and poly(ADP-ribose)polymer (PARpolymer) formation
in PC12 cells.16 Exposure to 150 µM H2O2 for 3 h
produced cell damage, as evaluated by MTT assay,17 and
the damage was significantly attenuated by pretreat-
ment (1 h prior to H2O2 exposure) with 32 at 10-8-10-5
M in Figure 3. Figure 4 shows the effect in a mouse
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
model of Parkinson’s disease (PD).18 The four-dose
paradigm of MPTP intoxication induces severe cell
injury. 32 was orally administrated twice to C57BL/6
mice (3.2, 10, 32 mg/kg, po) 1 h prior to the first and
third injections of MPTP, and after 4 days, each stria-
tum was dissected to quantify dopamine (DA) and its
metabolites dihydroxyphenylacetic acid (DOPAC) and
homovanillic acid (HVA). In this model, pretreatment
with 32 significantly prevented the depletion of striatal
DA, DOPAC, and HVA contents. 32 had no monoamine
oxidase-B (MAO-B) inhibitory activity and no dopamine
transporter (DAT) binding affinity, suggesting that 32
was not able to affect MPTP metabolism or inhibit the
accumulation of MPTP metabolites into the cells. Fur-
thermore, 32 had no CNS toxicity in a preclinical study.
Detailed pharmacological properties and the neuro-
protective effects of our PARP inhibitors will be pub-
lished in due course.16
brain (µg/g tissue)
plasma (µg/ml)
ratio
0.5 h:
2.0 h:
5.6 ( 2.30
2.1 ( 1.00
1.0 ( 0.41
0.5 ( 0.21
5.6
4.2
Metabolism by Liver Microsomes in Vitrod
n ) 3, CLint (mL/min/kg)
mouse
2250 ( 104
rat
dog
human
156 ( 9.5
1236 ( 69
141 ( 13
Administration as the HCl salt. BA ) bioavailability, c The
a
b
results were shown as the mean ( SE. The concentration was
measured at 0.5 and 2.0 h by HPLC after po administration at 32
d
mg/kg in mice. 32 was incubated at 37 °C with liver microsomes
from mice, rats, dogs, and humans in the presence of the NADPH-
generating system; see Supporting Information.
derivatives is outlined in Table 4. In general, substitu-
tion at the para position of the phenyl ring was more
advantageous to activity than either meta or ortho
substitution. A 2-fold improvement in activity was
obtained by p-fluoro 19, p-methoxy 25, and p-cyano 29
with IC50 ) 8.9, 8.3, and 6.0 nM, respectively.
After our SAR examination and extensive research,
we selected 32 as a potential candidate, since it inhib-
ited PARP-1 activity with an IC50 of 13 ( 1.1 nM (mean
( SE, n ) 4) and was orally active with high brain
penetration. Table 5 shows the pharmacokinetic profiles
in mouse, rat, and dog. There was more improved
bioavailability in the dog for the HCl salt of 32 (70%
oral bioavailability with a 4.8 h iv half-life) than in other
species, which is explained by metabolic stability as
shown by measurement of in vitro clearance using liver
microsomes. The concentrations of 32 in plasma and
brain were measured by HPLC after oral administration
We have discovered potent PARP-1 inhibitors accord-
ing to a rational discovery strategy, employing random
screening to find the seed, structure-based drug design
by modeling to generate the lead, classical SAR and

4154 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Letters
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combinatorial chemistry to optimize the lead, and X-ray
analysis to validate structure conformation. The newly
designed PARP-1 inhibitor 32 exhibited potent inhibi-
tory activity in vitro and in vivo with neuroprotective
activity and good bioavailability with high brain pen-
etration. These findings suggest that 32 could be an
attractive therapeutic candidate for neurodegerative
disorders such as Parkinson’s disease.
Ack n ow led gm en t. We express our thanks to Dr.
Kazuo Sakane and Dr. Masayuki Kato for their practical
guidance and Dr. David Barrett for his critical reading
of the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Typical experiment
details and characterization for compounds 3-32, X-ray data
for 1, and description of the in vitro PARP inhibitory assay,
ev vivo assay, and in vitro metabolism. This material is
available free of charge via the Internet at http://pubs.acs.org.
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