Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a tetrahydro-1(2H)-isoquinolinone Scaffold: Synthesis, biological evaluation and X-ray crystal structure

Article abstract of DOI:10.1055/s-2005-865324

The synthesis, activity and physical properties of two series of novel potent tetrahydro-1(2H)-isoquinolinone based PARP-1 inhibitors are described. The new structural classes with a non-planar ring system interact specifically with the PARP-1 protein at the nicotinamide-binding site. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2005-865324

1550
PAPER
Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors Based on a
Tetrahydro-1(2H)-isoquinolinone Scaffold: Synthesis, Biological
Evaluation and X-ray Crystal Structure
New
PARP-1
tInhibit
e
o
f
n
a
Tetra
a
hydro-1(2
H
n
)
-isoquinolinone
SP
caffold eukert,*1 Uwe Schwahn, Stefan Güssregen, Herman Schreuder, Armin Hofmeister
Aventis Pharma Deutschland GmbH, a company of the sanofi-aventis group, TD Cardiovascular, TD Thrombosis and Angiogenesis and
Chemical Sciences, 65926 Frankfurt, Germany
Fax +49(69)331399; E-mail: armin.hofmeister@sanofi-aventis.com
Received 23 December 2004
Dedicated to Professor Bernd Giese on the occasion of his 65th birthday
amide carbonyl group to be in the anti-conformation 1a
Abstract: The synthesis, activity and physical properties of two se-
that is in equilibrium with the syn-conformation 1b. The
first PARP inhibitors identified, such as 3-aminobenz-
ries of novel potent tetrahydro-1(2H)-isoquinolinone based PARP-
1 inhibitors are described. The new structural classes with a non-
amide (2), are close structural analogues to the nicotina-
mide moiety of NAD+.7 However, this compound lacks
good potency, which can be attributed to the conforma-
tional flexibility of the carboxamide carbonyl group.
Hence, restricting this carbamoyl moiety to the anti-con-
formation by formation of an adjacent ring led to new gen-
erations of compounds as shown in the isoquinolinone 3,
planar ring system interact specifically with the PARP-1 protein at
the nicotinamide-binding site.
Key words: medicinal chemistry, PARP inhibitors, structure-activ-
ity relationship, isoquinolinones, crystal structure
Introduction
5[H]phenanthridin-6-one
4
or
dihydrocyclopen-
ta[lmn]phenanthridin-5-one 5 series which were more po-
Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30),
the most prominent member of the PARP family, is a
DNA-binding protein activated by nicks in DNA occur-
ring during inflammation, ischemia, neurodegeneration or
cancer therapy.2 Activated PARP-1 consumes NAD+ that
is cleaved into nicotinamide and ADP-ribose and poly-
merizes the latter onto nuclear acceptor proteins. This pro-
cess is pivotal for the maintenance of genomic stability.
When over-activated, however, PARP-1 can cause exten-
sive polymerization of ADP-ribose, leading to the deple-
tion of NAD+ and, subsequently, a decrease in the level of
intracellular ATP. This depletion of ATP can culminate in
cell dysfunction and cell death through a necrotic path-
way.3 Studies done with PARP-1 deficient mice demon-
strated that a lack of PARP-1 renders them resistant to
cerebral ischaemia.4 This observation was supplemented
by recent work with potent inhibitors demonstrating the
beneficial effects of PARP inhibition in animal models of
stroke, heart ischemia and cancer.5 Therefore, PARP-1 is
regarded as a promising target for the development of
drugs useful in various forms of inflammation, ischemia-
reperfusion injury and as an adjunct in cancer therapy.
tent by several orders of magnitude7,8 (Figure 1).
The overwhelming majority of known PARP inhibitors
bind to the nicotinamide-binding site, where they act as
competitive inhibitors to NAD+. As depicted in Figure 1,
the carboxamide moiety of nicotinamide forms three hy-
drogen bonds with the catalytic domain of PARP-1; one to
the hydroxyl group of Ser904 and two to the amide back-
bone of Gly863.6 This binding mode requires the carbox-
Figure 1 Structural requirements for PARP inhibition and classes
of PARP-1 inhibitors
Despite this improvement in potency, clinical develop-
ment is still slow which might be attributed to safety is-
sues. As mentioned above, most PARP inhibitors have
extended planar structures that can have DNA-intercalat-
ing properties.9 Therefore, inhibitors with less planar
SYNTHESIS 2005, No. 9, pp 1550–1554
x
x
.
x
x
.2
0
0
5
Advanced online publication: 19.04.2005
DOI: 10.1055/s-2005-865324; Art ID: C05404SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
New PARP-1 Inhibitors Based on a Tetrahydro-1(2H)-isoquinolinone Scaffold
1551
structures, which should be devoid of this unwanted prop- wave conditions as shown in Scheme 2. Deprotection of
erty are desirable. Herein, we describe substituted 5,6,7,8- the pyridone functionality with chlorotrimethylsilane and
tetrahydro-1(2H)-isoquinolinones 6 as PARP-1 inhibi- potassium iodide furnished cleanly the analogues 6a–i.
tors. This class of compounds was designed to overcome
the planar ring system found in known PARP-1 inhibitors
albeit maintaining their good potency.
Chemistry
The synthesis of the key building blocks 12 is outlined in
Scheme 1. The preparation of the methyl-substituted
compound 12b started with phthalic anhydride (7), which
was heated with ethyl 2-aminopropionate to yield the
phthalimidopropionate 8 in 89% yield. Phthalimido ester
Scheme 2 Reagents and conditions: a) aniline, t-BuONa, toluene,
Pd2dba3, t-Bu2P-biphenyl, 150 °C, microwave; b) TMSCl, KI,
MeCN, 60 °C
rearrangement in the presence of sodium methoxide af-
Synthesis of the furyl substituted compounds 6j–u origi-
forded the hydroxyisoquinolinone 9 which was reduced to
nated from bromides 12 as well. Suzuki coupling using
isoquinolinone 10b in hydroiodic acid in the presence of
the Fu conditions furnished aldehydes 14.11 The requisite
5-formylfuran-2-boronic acid is either commercially
red phosphorus in moderate yield after extended periods
of time.10 Hydrogenation of 10b in acetic acid at 5 bar in
available or can be prepared by an excellent protocol from
the presence of platinum resulted in 5,6,7,8-tetrahydroiso-
quinolinone 11b which could be easily isolated by recrys-
tallization from methanol in 83% yield. In the case of the
commercially available isocarbostyril (10a) the hydroge-
nation occurred less selectively and provided next to the
desired 11a (60% yield) some 3,4-dihydro-2H-isoquino-
lin-1-one, which could be removed by recrystallization
from water. Both compounds were excellent substrates for
bromination in 4-position: 11a was brominated with bro-
mine in chloroform whereas 11b reacted best with N-bro-
mosuccinimide in methanol. The intermediates were
subsequently O-methylated with methyl iodide in the
the patent literature.12 Deprotection with chlorotrimethyl-
silane and potassium iodide yielded aldehydes 15, which
were subjected to reductive amination employing poly-
mer-bound triacetoxyborohydride in THF. The use of the
polymer-bound reducing agent facilitated the work-up
procedure and allowed better recovery of the water-solu-
ble analogues 6j–s. Compound 6t (R1, R3 and R4 = H) was
prepared by reductive amination of 15a with sodium boro-
hydride in the presence of a large excess of ammonium ac-
etate. This compound served as starting material for the
preparation of analogue 6u by acylation with nicotinoyl
chloride.
presence of silver carbonate to afford 12a and 12b, re-
spectively. The transformation into the annulated meth-
oxypyridines proved necessary, as palladium-catalyzed
reactions did not proceed with the brominated 5,6,7,8-tet-
Results and Discussion
rahydroisoquinolinones. Compounds 6 substituted with
1
All compounds were fully characterized by H NMR
anilines could be prepared in moderate yields by Hartwig–
spectroscopy, high resolution MS and HPLC and were
Buchwald amination from bromides 12 employing micro-
tested as inhibitors for human PARP-1 (see experimental
section). Solubility was determined in phosphate buffered
saline at pH 7.4 (24 h equilibrium). Two series of potent
4-substituted tetrahydro-1(2H)-isoquinolinones were
identified, namely the aniline substituted compounds
(Table 1, 6a–i) and the furyl substituted compounds
(Table 2, 6j–u) (Scheme 3). Table 1 shows the structure–
activity relationship (SAR) for a series of simple substitu-
ents R1 and R2. Compounds with a methyl group at the R1
position (6a,c) proved to be 3–7 fold more potent than the
corresponding analogues derivatives with hydrogen at
this position (6g,h). A possible explanation for this could
be that the methyl group at R1 position has a favorable en-
tropical effect by restricting the conformations of the
aniline moiety. The most active compounds 6a and b both
have a R2 substituent with a carbonyl functionality indi-
Scheme 1 Reagents and conditions: a) ethyl 2-aminopropionate,
120 °C, 89%; b) NaOMe, MeOH, reflux, 63%; c) red P, 55% HI,
160 °C, 46%; d) PtO2, H2 (3–5 bar), AcOH, 60–83%; e) 12a: Br2,
CHCl3, 5 °C, 96%; 12b: NBS, MeOH, 0 °C, 84%; f) MeI, Ag2CO3,
cating a strong and specific interaction with the enzyme-
binding site. This hypothesis was later verified by an in-
hibitor/enzyme crystal structure with 6a (see next sec-
tion). Compound 6d with the inverted amide functionality
CHCl3, 50 °C, 83–84%
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1552
S. Peukert et al.
PAPER
Table 2 Structure–Activity Relationship for Compounds 6j–u
Bearing a Furyl Substituent
Table 1 Structure–Activity Relationship for Compounds 6a–i
Bearing an Aniline Substituent
R1 R3/R4
IC50 Sol.
R1
R2
IC50 (mM)
(mM) (mg/mL)a
6a
6b
6c
6d
6e
6f
Me
Me
Me
Me
Me
Me
H
COMe
0.09
0.08
0.30
0.14
1.21
0.61
0.69
0.80
1.43
6j
6k
6l
Me -(CH2)4-
Me H, Bu
0.30 2.40
0.24 n.d.
CONMe2
SO2Me
NHCOMe
OCF3
Me H, 1-methylpyrazol- 0.33 3.40
4-ylmethyl
6m
6n
6o
H
H
H
-(CH2)4-
H, Bu
0.11 1.97
0.25 0.82
CH2OH
COMe
H, 1-methylpyrazol- 0.18 n.d.
4-ylmethyl
6g
6h
6i
H
SO2Me
6p
6q
H
H
H, CH2Ph
0.15 n.d.
H
1-methylimidazol- 2.76 n.d.
2-ylmethyl, CH2Ph
6r
6s
H
H
H, (CH2)2NMe2
0.46 1.41
H, pyridin-3-
ylmethyl
0.99 12.18
6u
H
H, nicotinoyl
0.79 n.d.
presumably can still develop the same interaction albeit
with slightly decreased strength. The same seems to be
true for compound 6c with a methanesulfonyl substituent
at this position. Compounds that lack a hydrogen acceptor
at this position (e.g. 6e) showed diminished potency.
a n.d.: Not determined.
Compound 6f regained some of the potency as the oxygen and the corresponding unsubstituted compounds 6m–o is
of the hydroxylmethyl group might serve as acceptor in- small with a slight preference for the unsubstituted com-
stead of a carbonyl oxygen. Attempts to further increase pounds. The SAR regarding R3/R4 is somewhat flat as
the potency by restricting the rotational freedom of the shown in examples 6m–p and r. Secondary and primary
carbonyl group by incorporating it into a ring (6i vs 6g) aliphatic, heteroaromatic, aromatic and basic functional-
were not successful, indicating the sensitivity of the inter- ized side chains were all tolerated, indicating that this part
action between this functionality and the binding site. of the molecule is not involved in specific interactions
Compound 6a was further profiled and showed next to the with the binding site. As seen from the X-ray structure of
IC50 value of 90 nM a low inhibitory constant (Ki = 54 compound 6j (see next section), the main interaction at
nM) and EC50 value in the cell-protection assay of 0.2 mM.
this portion of the molecule is between the positively
charged nitrogen and a conserved water molecule in the
binding site. However, steric bulk is of importance as
demonstrated with compound 6q. This tertiary amine with
In the furyl series the difference in potency between com-
pounds 6j–l with a methyl substituent at the R1 position
Scheme 3 Reagents and conditions: a) 5-formylfuran-2-boronic acid, Pd2dba3, KI, (t-Bu)3PHBF4, THF, 60 °C, 79–100%; b) TMSCl, KI,
MeCN, 60 °C, 27–55%; c) macroporous BH(OAc)3 or NaCNBH3, THF, amine; d) nicotinoyl chloride, Et3N, CH2Cl2
Synthesis 2005, No. 9, 1550–1554 © Thieme Stuttgart · New York

PAPER
New PARP-1 Inhibitors Based on a Tetrahydro-1(2H)-isoquinolinone Scaffold
1553
two larger substituents R3/R4 showed clearly diminished chain of His862. The N of the pyridone ring interacts with
potency compared to smaller secondary amines 6o or p. the carbonyl oxygen of Gly863, while the carbonyl oxy-
Interestingly, pyridyl substituted compounds 6s and u ex- gen of the pyridone interacts with the main chain N of the
hibited comparable activity although 6u with an amide same Gly863. In addition, this carbonyl O makes also a
functionality cannot form the interaction described for the hydrogen bond with the hydroxyl side chain of Ser904.
amine functionality. Modeling studies suggest that in this The annulated cyclohexyl ring forms optimal van der
case the pyridyl nitrogen of 6u forms a weak hydrogen Waals contacts with the protein and occupies the same
bond to the Arg878 main chain nitrogen that compensates space as the benzene ring in other bicyclic lactam inhibi-
for the loss of the interaction of the amine functionality tors. Modeling studies suggest that this is the position of
mentioned above.
the aromatic part of the nicotinamide ring of the natural
substrate NAD+.6
Compounds 6j–u typically showed excellent solubilities
in aqueous solution at pH 7.4 which make them readily The 3-acetylphenyl group of 6a is oriented almost perpen-
suitable for intravenous applications. Moreover, com- dicular to the pyridone ring. The carbonyl oxygen displac-
pound 6j exhibited an inhibitory constant of 15 nM and an es a water molecule and interacts with the main-chain
EC50 value of 0.33 mM in the cell-protection assay com- nitrogen of Tyr896, which can explain the high potency of
pared to the free enzyme IC50 value of 0.30 mM, which compounds 6a and b.
demonstrates its good cell permeability.
The furan ring of 6j is also almost perpendicular to the py-
ridone ring at about the same position as the 3-acetylphe-
nyl group of 6a. However, the pyrrolidine ring is at a
position about 90° away from the position of the acetyl
Structural Data
substituent of 6a.The pyrrolidine nitrogen is involved in a
The observed SAR can be explained with the aid of two
strong hydrogen bond to a bound water molecule, which
crystal structures of the active fragment of chicken PARP-
is held in place by hydrogen bonds to the side chains of
1 in the presence of the aniline substituted inhibitor 6a and
Gln763 and Glu766 and another water molecule in the
network of water molecules in the PARP active site. We
the furan-substituted inhibitor 6j (Figure 2a,b).13
Both inhibitors are bound in the donor NAD+ site, as iden- did not observe any further specific interactions between
tified by Ruf et al.14 The N-terminal domain in our struc- either the furan ring or the pyrrolidine ring and the pro-
tures is slightly rotated with respect to the PARP tein.
structures available in the protein data bank (PDB), lead-
ing to an average root-mean-square displacement of about
1.5 Å for this domain. At present it is not possible to de-
In conclusion, we have discovered and optimized substi-
tuted tetrahydro-1(2H)-isoquinolinones as a new class of
PARP-1 inhibitors. Apart from high potency, compounds
termine whether it is due to our particular crystallization
with excellent solubility could be identified and their
conditions (r.t. vs. 4 °C), or whether the inhibitors induce
binding conformation has been evaluated with X-ray crys-
tal structures of the ligands and the PARP-1 protein. The
this conformational change.
The binding mode of the tetrahydroisoquinolinone ring use of these non-planar inhibitors which could be advan-
system is the same for both inhibitors. The pyridone ring tageous in terms of non-mechanism based side effects is
is stacked between the aromatic side chain of Tyr907, the currently studied in vivo.
main-chain atoms of Tyr896 and Phe897 and the side
Figure 2a (left): Crystal structure of compound 6a and cPARP-1 protein;
Figure 2b (right): Crystal structure of compound 6j and cPARP-1 protein
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S. Peukert et al.
PAPER
PARP Enzyme Inhibition Assay
References
The inhibitory potency of substances was measured in an enzymatic
assay using recombinant human PARP-1 protein. In a 50 mL total
reaction volume, 2 mg/mL enzyme were incubated with different
concentrations of test substance in a buffer containing Tris (50
mM), MgCl2 (5 mM), dithiothreitol (1 mM), NAD (200 mM), triti-
ated NAD (0.1 mCi/mL), sheared DNA (0.1 mg/mL) and histone
(0.1 mg/mL) at pH 8.0. After 1 h incubation at r.t., the reaction was
stopped by adding aq 20% trichloroacetic acid (150 mL) and incu-
bated on ice for 10 min. Protein precipitates were collected on glass
fiber filters, washed three times with aq 20% trichloroacetic acid
and incorporated radioactive poly(ADP-ribose) polymer was mea-
sured in a Wallac Trilux Microbeta by liquid scintillation counting.
IC50 values were determined as the substance concentration at
which precipitable radioactivity reached 50% of the value attainable
by incubation of the reaction mixture with solvent only. Inhibitory
constants (Ki) of the test substances were determined with the same
enzyme reaction as described above, only that different concentra-
tions of the substrate NAD were used and the incubation time was
reduced to 10 min. Ki values were determined by linear regression
assuming a purely competitive mechanism of inhibition.
(1) New address: S. Peukert, Novartis Institutes for Biomedical
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(13) The catalytic fragment of chicken PARP-1 was obtained
from Prof. DeMurcia of the Ecole Supérieure de
Biotechnology de Strasbourg. Native PARP-1 was
crystallized at pH 8.5 with 18% PEG 4000 and 8% i-PrOH
as precipitant, following the protocol of Jung et al.: Jung, S.;
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114. Inhibitor complexes were obtained by soaking native
crystals with the appropriate inhibitors. The space group and
cell dimensions of the crystals obtained were the same as in
the published crystal structures. We obtained well
diffracting crystals, diffracting to 2.2 and 2.1 Å resolutions,
respectively. We did not observe any major differences
between our PARP structures and the PARP structures
present in the PDB.
ATP Consumption Assay in Cardiomyoblasts
The protective potency of the substances was measured in a cellular
model of cytotoxic injury. Rat cardiomyoblasts (H9c2) were seeded
at 40,000 cells per well in a 96-well plate and incubated overnight
in RPMI1640, 10% fetal calf serum. Then cells were pretreated for
15 min with different concentrations of the test substance and after
adding H2O2 (300 mmol) the cells were lyzed for 1 h and ATP con-
tent determined by luciferase reaction. The half-maximal effective
concentration (EC50) of the substances was determined as the con-
centration at which the ATP content of injured cells reached 50% of
the level that could maximally be attained by the same compound at
higher substance concentrations.
Acknowledgment
We wish to thank Horst Plankenhorn and Michael Schnierer for
their technical assistance in the synthesis of the compounds and also
Antje Schlüter for her assistance in the biological evaluation of the
compounds. We also thank Alexander Liesum, Volker Brachvogel
and Petra Lönze for excellent technical assistance with the crystal-
lization, data collection and refinement of the structures.
(14) Ruf, A.; De Murcia, J. M.; De Murcia, G. M.; Schulz, G. E.
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Synthesis 2005, No. 9, 1550–1554 © Thieme Stuttgart · New York