Identification of substituted pyrazolo[1,5-a]quinazolin-5(4H)-one as potent poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.05.113

A novel series of pyrazolo[1,5-a]quinazolin-5(4H)-one derivatives proved to be a potent class of PARP-1 inhibitors. An extensive SAR around the 3-position of pyrazole in the scaffold led to the discovery of amides derivatives as low nanomolar PARP-1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.05.113

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4196–4200
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of substituted pyrazolo[1,5-a]quinazolin-5(4H)-one as potent
poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors
*
Federica Orvieto , Danila Branca, Claudia Giomini, Philip Jones, Uwe Koch, Jesus M. Ontoria,
Maria Cecilia Palumbi, Michael Rowley, Carlo Toniatti, Ester Muraglia
IRBM - Merck Research Laboratories Rome, Via Pontina km 30,600, Pomezia, 00040 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrazolo[1,5-a]quinazolin-5(4H)-one derivatives proved to be a potent class of PARP-1
inhibitors. An extensive SAR around the 3-position of pyrazole in the scaffold led to the discovery of
amides derivatives as low nanomolar PARP-1 inhibitors.
Received 29 April 2009
Revised 27 May 2009
Accepted 27 May 2009
Available online 2 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PARP-1 inhibitor
Pyrazole
O
Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nucle-
ar enzyme, discovered four decades ago, with an important role in
the cellular life cycle.1 It is involved in many fundamental pro-
cesses including DNA repair, transcriptional regulation as well as
governing cell death.2 The activation of PARP-1 occurs upon bind-
ing of the enzyme to single or double strand DNA breaks, causing
the formation of long and branched poly(ADP-ribose) polymers
using nicotinamide adenine dinucleotide (NAD+) as substrate.
PARP-1 catalyzes the addition of these long branched chains onto
itself and on other target proteins acceptors.3 These PAR chains
serve to recruit other DNA repair machinery to the site of damage,
and the DNA breaks are thereafter repaired by base excision repair
(BER). The role of PARP-1 in the regulation of DNA integrity has
made it an attractive target for cancer therapy. Indeed, PARP-1
inhibitors have been shown to sensitize cells to cytotoxic agents
such as temozolomide, topoisomerase inhibitors and cisplatin, as
well as ionizing radiation.4 Moreover several recent articles have
demonstrated that cells deficient in BRCA1 and BRCA2 proteins, in-
volved in the repair of DNA double-strand breaks by homologous
recombination, are highly sensitive to PARP-1 inhibitors.5 Several
PARP-1 inhibitors are currently being explored in the clinic as
mono- and combination therapy in cancer patients.6
O
X
NH2
NH
R
1 R = NH2 or OH
2a (X = N)
2b (X = CH)
O
O
NH
NH
A
B
R2
N
N
C
R1
3
4
Figure 1. Structure of PARP-1 inhibitors.
studies, research focused on the design of PARP-1 inhibitors in
which the carboxamide group could be locked into the required
conformation. This was achieved with ring closure of the carbox-
amide into bicyclic systems. Bicyclic lactams such as quinazolinon-
es (2a, Fig. 1) and isoquinolinones (2b) are two example of ‘first
generation’ PARP-1 inhibitors.9 Further development of these bicy-
clic systems is represented by the related tricyclic lactam 3. Elabo-
ration of these core structures in order to enhance potency,
improve pharmacokinetic properties and increase aqueous solubil-
ity is reported in literature.10
The first PARP-1 inhibitors identified (3-substituted benzam-
ides 1, Fig. 1) are structural analogues of the nicotinamide moiety
of NAD+.7 However these compounds have a low potency and spec-
ificity. Further studies revealed that restriction of the carboxamide,
which is normally free to rotate, to the biologically-active anti-con-
formation provided an increase in binding affinity.8 Based on these
* Corresponding author. Tel.: +39 06 91093277; fax: +39 06 91093654.
E-mail address: federica_orvieto@merck.com (F. Orvieto).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.113

F. Orvieto et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4196–4200
4197
We envisaged the possibility to develop novel tricyclic lactam
compounds as potential PARP-1 inhibitors by replacement of the
C ring of 3 with a 5-member nitrogen containing heterocycle. Here
we report the synthesis and biological evaluation of cyclic pyrazole
derivatives 4, in which one of the pyrazole nitrogens is embedded
in the quinazolinone 2a. The rational for this was based on their
synthetic accessibility, as they could be readily prepared, and on
the potentially improved physiochemical properties as a result of
the inclusion of the weakly basic pyrazole motif.
of the tricyclic system. The results in PARP-1 inhibition assay are
summarized in Table 1.
Replacement of the methyl group of 5 with higher alkyls, such
as i-butyl (6) and cyclohexyl (7) gave compounds basically equipo-
tent with 5. When a phenyl substituent was introduced in position
2 of the tricyclic system (8) a 3-fold boost in activity was achieved,
displaying an IC50 = 0.1
lM. The isomeric phenyl derivative (4,
R1 = H, R2 = Ph) was inactive (IC50 > 1
lM) so no further isomeric
derivatives were explored. The effects of further substitutions on
the phenyl ring of 8 were investigated. 4-methyl (9) and 4-ethyl
(10) substitutions provided 5-fold less active compounds with
respect to 8, while 4-propyl and 4-t-butyl derivatives displayed
a marked drop in PARP-1 potency (11 and 12). Similarly, a
drop in activity was observed also by the 3- and 4-fluoro deriva-
tives (13 and 14), while the other halogen substitutions
The first tricyclic compound tested in the PARP-1 enzyme assay
to probe the hypothesis was the commercially available methyl
derivative 511 which, pleasingly, showed good activity
(IC50 = 0.3
erature, IC50 = 0.52
l
M) comparable to the (5H) phenanthridin-6-one (3, lit-
l
M).10a This encouraging result prompted us to
target a library of analogs with different substituents in position 2
Table 1
Table 2
SAR study on 2-position of the pyrazolo[1,5-a]quinazolin-5(4H)-one
SAR study on meta and para position of 8
O
NH
O
N
N
R
NH
N
N
a,b
R1
Compd
R
PARP-1 IC50
(lM)
5
6
Me
0.3
0.4
R2
iBu
7
8
9
Cyclohexyl
Ph
0.2
0.1
0.5
0.5
>1.0
>1.0
>1.0
>1.0
0.5
0.5
0.6
0.7
>1.0
>1.0
a
Compd
R1
H
R2
H
PARP-1 IC50
0.1
(lM)
4-Me–Ph
4-Et–Ph
4-Pr–Ph
4-tBu–Ph
3-F–Ph
4-F–Ph
3-Cl–Ph
4-Cl–Ph
3-Br–Ph
4-Br–Ph
4-OCF3–Ph
4-CN–Ph
8
10
11
12
13
14
15
16
17
18
19
20
O
N
N
27
28
H
H
1.0
0.8
O
N
N
OMe
21
22
23
0.2
0.1
0.2
OMe
O
N
29
30
H
0.5
1.4
OMe
OMe
N
OMe
O
N
N
H
H
OMe
OMe
O
OMe
OMe
24
25
0.03
N
N
31
1.3
2.5
S
0.15
0.2
O
N
O
32
H
26
N
a
Values are means of two experiments. SD is 30%.
See Ref. 17 for PARP-1 inhibition assay.
a
b
See note a–b in Table 1.

4198
F. Orvieto et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4196–4200
(15–18) provided compounds with an IC50 ranging between 0.5
and 0.7 M. Likewise, different substitutions such as trifluoro-
methoxy (19) and cyano (20) in para position proved to be not
low nanomolar inhibitor, displaying IC50 = 0.03 lM, and to be 3-
l
fold more active then the parent phenyl derivative 8. Introduction
of phenyl bioisosters such as thiophene and furan (25 and 26) re-
tained the PARP-1 activity of the phenyl analog 8.
tolerated.
Interestingly, the presence of the electron-donating groups was
tolerated for PARP-1 activity as demonstrated by compounds 21–
23. In particular, the trimethoxy compound 24 proved to be a
It is widely reported that most of the tricyclic unsaturated
PARP-1 inhibitors show limited solubility in both organic and
Table 3
SAR of 5-oxo-4,5-dihydropyrazolo[1,5-a]quinazoline-2-carboxamides
O
NH
N
N
R
O
a
Compd
R
PARP-1 IC50
0.3
(lM)
33
N
N
N
N
34
35
0.3
0.6
Figure 2. Model of 37 in the NAD binding site of PARP enzyme (pdb code: 2pax).
Derivative 37 was manually placed in the NAD binding site and, to obtain the
minimum conformation, a conformational analysis was performed using the MMFF
forcefield18 as implemented in Macromodel 7.0.19
N
N
N
N
36
37
0.1
Table 4
N
SAR of 5-oxo-4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxamides
0.01
O
N
NH
R
N
N
38
39
0.03
0.7
O
N
NH
a
Compd
R
PARP-1 IC50
>1.0
(lM)
N
O
N
46
N
N
N
47
48
>1.0
N
N
N
40
1.1
N
O
N
>1.0
>1.0
41
42
0.07
0.08
NH
NH
N
O
N
49
N
NH
NH
H
a
See note a–b in Table 1.
43
44
0.14
0.09
0.14
N
N
O
O
O
O
NH
NC
R
OH
a
NH
+
O
NH
NH2
N
N
R
HCl
N
O
45
50
51
6-26
N
H
Scheme 1. Synthesis of derivatives in Table 1. Reagents and conditions: (a) acetic
acid, MW, 150 °C, 5 min. Yield 30–65%.
a
See note a–b in Table 1.

F. Orvieto et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4196–4200
4199
OEt
OEt
O
O
N
N
O
NH
NH
53
CONR1R2
b,c
CO2Et
N
O
N
N
OH
a
55
46-49
NH
NH2
HCl
O
O
N
50
NH
NH
b,c
N
CO2Et
ONa
N
N
N
CONR1R2
CO2Et
54
56
33-45
Scheme 2. Synthesis of carboxamides in Tables 3 and 4. Reagents and conditions: (a) DMF, MW, 140 °C, 105 min; (b) 2 M NaOH, MeOH, MW, 90 °C, 5 min; (c) PS-DCC, HOBt,
DMF, R1R2NH, rt, 60 min.
aqueous solvents, probably due to the planarity of the tricyclic
core.12 In an attempt to improve solubility while maintaining or
possibly increasing potency, we thought to introduce polar amides
in the meta and para positions of 8, bearing a basic amino function-
ality as solubilizing group.
As shown in Table 2, piperazine compound 27 caused a 10-fold
drop in activity compared to 8 (1.0 lM vs 0.1 lM). Also open ana-
logue 28 and fused bicyclic piperazine 29 proved to be less potent
with respect to the unsubstituted phenyl. The detrimental effect of
the introduction of an amide substituent on the phenyl ring was
even more evident in the 4-position, that produced compounds
inhibitory activity (Table 4), in line with the result on the 3-phenyl
analogue.
The 2-substituted pyrazoloquinazolone derivatives in Table 1
were prepared according to the literature as described in Scheme
1.13 The tricyclic pyrazoloquinazolone amides in Tables 3 and 4
were obtained by derivatization of the corresponding carboxylic
acids. The tricyclic esters 5514 and 5615 were synthesized as de-
scribed in Scheme 2. Condensation of benzoic acid hydrazide (50)
with ethyl 2-cyano-3-ethoxyacrylate (53) or with sodium 1-cya-
no-3-ethoxy-3-oxoprop-1-en-2-olate (54), respectively, under
microwave irradiation provided esters 55 and 56. Hydrolysis with
NaOH under microwave irradiation followed by amide coupling of
the resulting carboxylic acid with the appropriate amines, using
polymer supported carbodiimide as coupling reagent, led to the fi-
nal carboxamides (33–45 and 46–49).16
In conclusion, we have reported the synthesis and biological
evaluation of a new class of tricyclic lactam PARP-1 inhibitors con-
taining a pyrazole ring fused to the isoquinolinone (4, Fig. 1). SAR
at the 2-position of the tricyclic system with alkyl and phenyl sub-
stituents provided submicromolar inhibitors (24, IC50 = 30 nM).
Introduction of polar amides in position 2 of the tricyclic lactam
led to the identification of derivatives such as 37 as low nanomolar
PARP-1 inhibitors (IC50 = 10 nM).
with an IC50 > 1.0 lM (see 30–32, Table 2).
Based on the above disappointing results we envisaged the pos-
sibility of a steric restriction in that region of the molecule so we
thought to introduce the polar amide groups directly linked to
the tricyclic core (R1 and R2 in Fig. 1, structure 4), removing the
phenyl spacer.
Interestingly, substitutions at the C-2 position resulted in sub-
micromolar inhibitors: substituted piperazines 33 and 34 (see
Table 3) displayed IC50 = 0.3 lM against PARP-1, comparable to
the lead compound 5 bearing a single methyl. The fused bicyclic
piperazine 35 showed lower activity, while the open analogue 36
proved to be 3-fold more active than 33. A further improvement
in activity was achieved with the diazepane derivatives 37 and
38 which were low nanomolar PARP-1 inhibitors (IC50 = 10 and
30 nM, respectively), resulting to be the most potent of this class
of compounds, presumably due to a beneficial interaction of the
amine with acid residues of the protein. Modeling studies of 37
suggest that Asp766, which is close to the basic amine, can change
its conformation in order to maximize its charge/charge interac-
tions with the ligand, and thus contribute to binding (Fig. 2). Deriv-
ative 37 was tested for its ability to inhibit the formation of PAR
polymers in HeLa cells upon stimulation of DNA damage with
References and notes
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H2O2 where it showed an EC50 = 3.9 lM.
Alkylated piperidines 39 and 40 were only moderately active,
while good potency was achieved with methyl piperidine (41) and
methyl pyrrolidine (42) substitutions. Aliphatic derivatives 43, 44
and 45 showed comparable good potency indicating that both the
branching and the length of the spacer between the amide nitrogen
and the basic nitrogen did not dramatically affect the activity,
although all three were less active than diazepanes 37 and 38.
Taking into account the above results, substitutions in the 3-po-
sition of tricyclic system were also explored. Installation of the best
amide fragments previously described dramatically diminished the
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18. Synthesis of 56: A 0.5 M solution of 2-hydrazinobenzoic acid hydrochloride in
DMF was treated with sodium l-cyano-3-ethoxy-3-oxoprop-l-en-2-olate
(1 equiv). The reaction mixture was heated at 140 °C under microwave
irradiation for 105 min and then poured into water. The resulting solid was
isolated and dried under vacuum (yield = 66%). 1H NMR (300 MHz, DMSO-d6,
300 K) d 1.32 (t, J = 7.1 Hz, 3H), 4.34 (q, J = 7.1 Hz, 2H), 6.26 (s, 1H), 7.58 (t,
J = 7.5 Hz, 1H), 7.92 (t, J = 7.5 Hz, 1H), 8.10–8.20 (m, 2H), 12.34 (br s, 1H). MS m/
z (EI+): 258 (M+H)+.
19. As an example, synthesis of 38. A solution of 56 in MeOH 0.6 M was treated with
2 N NaOH (3.5 equiv). The reaction mixture was heated at 90 °C under
microwave irradiation for 5 min, then poured into water and acidified with
6 N HCl to pH 2. The solid formed was isolated to afford quantitatively 5-oxo-
4,5-dihydropyrazolo[1,5-á]quinazoline-2-carboxylic acid. 1H NMR (400 MHz,
DMSO-d6, 300 K) d 6.23 (s, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.87–7.97 (m, 1H), 8.10–
8.20 (m, 2H), 12.31(br s, 1H), 13.19 (br s,1H). MS m/z (EI+): 230 (M+H)+. To the
above carboxylic acid in DMF (0.1 M), HOBt (1.7 equiv) and PS-DCC (1.7 equiv)
were sequentially added. The resulting suspension was stirred at rt for 60 min,
then a solution of tert-butyl 1,4-diazepane-1-carboxylate (0.7 equiv) in DMF
was added and the reaction mixture was stirred at rt for 24 h. Filtration and
evaporation of volatiles provided Boc protected 38 that was purified by RP-
HPLC. Addition of TFA and evaporation afforded 38 (yield = 58%). 1H-NMR
(300 MHz, DMSOd6 + 2%TFA, 300 K) d 2.00–2.18 (m, 2H), 3.18–3.55 (m, 4H),
3.70 (t, J = 5.7 Hz, 1H), 3.80–3.87 (m, 1H), 4.0 (t, J = 5.7 Hz, 1H), 4.08–4.18 (m,
1H), 6.18 (d, J = 8.4 Hz, 1H), 7.48–7.58 (m, 1H), 7.85–7.92 (m, 1H), 8.03–8.10
(m, 1H), 8.14–8.20 (m, 1H), 8.76 (br s, 2H). MS m/z (EI+): 312 (M+H)+.
11. Compound commercially available from Bionet.
12. PARP-1 inhibition assay was conducted in buffer containing 25 mM Tris pH 8.0,
1 mM DTT, 1 mM Spermine, 50 mM KCl, 0.01 % Nonidet P-40 and 1 mM MgCl2.
PARP reactions contained 0.1
l
Ci [3H]-NAD (200,000 DPM), 1.5
l
M NAD+,
150 nM biotinylated NAD+, 1
l
g/mL activated calf thymus, and 1–5 nM PARP-
1. Auto reactions utilizing SPA bead-based detection were carried out in 50
volumes in white 96-well plates.
lL
Compounds were prepared in 11-point serial dilution in 96 well plate, 5
well in 5% DMSO/H2O (10Â concentrated). Reactions were initiated by adding
first 35 L of PARP-1 enzyme in buffer and incubating for 5 min at rt, then
10
L of NAD+ and DNA substrate mixture. After 3 h at rt these reactions were
terminated by the addition of 50 L Streptavidin-SPA beads (2.5 mg/ml in
200 mM EDTA pH 8). After 5 min, they were counted using TopCount
lL/
l
l
l
a
microplate scintillation counter. IC50 data was determined from inhibition
curves at various substrate concentrations.
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