Design and synthesis of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors. Part 3: In vitro evaluation of 1,3,4,5-Tetrahydro-benzo[c][1,6]- and [c][1,7]-naphthyridin-6-ones

Article abstract of DOI:10.1016/S0960-894X(03)00465-7

The 1,3,4,5-tetrahydro-benzo[c][1,6]- and [c][1,7]-napthyridin-6-ones are presented as a potent class of PARP-1 inhibitors. Derivatives of these partially saturated aza-5[H]-phenanthridin-6-ones were designed and synthesized with tertiary amines for salt

Full text of DOI:10.1016/S0960-894X(03)00465-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2513–2518
Design and Synthesis of Poly(ADP-ribose)polymerase-1
(PARP-1) Inhibitors. Part 3: In Vitro Evaluation of
1,3,4,5-Tetrahydro-benzo[c][1,6]- and [c][1,7]-naphthyridin-6-ones
Dana Ferraris,* Rica Pargas Ficco, Thomas Pahutski, Susan Lautar, Shirley Huang,
Jie Zhang and Vincent Kalish
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MA 21224, USA
Received 7 April 2003; revised 25 April 2003; accepted 6 May 2003
Abstract—The 1,3,4,5-tetrahydro-benzo[c][1,6]- and [c][1,7]-napthyridin-6-ones are presented as a potent class of PARP-1 inhibi-
tors. Derivatives of these partially saturated aza-5[H]-phenanthridin-6-ones were designed and synthesized with tertiary amines for
salt formation, thus enhancing aqueous solubility, ivformulation and their potential use in acute ischemic injuries (i.e., myocardial
ischemia and stroke). We found that partial saturation of the C-ring results in derivatives that are several times more potent than
the aromatic C-ring derivatives. The general synthetic routes are presented herein as well as thorough in vitro potencies and SAR
discussion for selected derivatives.
# 2003 Elsevier Ltd. All rights reserved.
Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30)
is an abundant nuclear enzyme with an important role
in the cellular life cycle.1,2 The PARP-1 enzyme has
three structural regions: the DNA binding domain con-
taining two zinc fingers, the automodification domain
and the catalytic domain.3 The catalytic domain is
responsible for converting nicotinamide adenine dinu-
cleotide (NAD+) into nicotinamide and adding an
ADP-ribose unit to the substrate.4 When over-activated,
PARP-1 can cause the consumption of NAD+ and
subsequently the depletion of intracellular ATP. This
depletion of ATP results in cell death through a necrotic
pathway and eventually results in ischemic tissue
damage.5,6 Recently, studies have demonstrated the uti-
lity of aqueous soluble PARP-1 inhibitors in rat models
of stroke and heart ischemia.7 These data indicate that
PARP-1 inhibitors could be utilized in a clinical setting to
treat ischemia-reperfusion injuries, an area of medicine
with unmet therapeutic needs.
whose structure–activity relationships with the enzyme
are well established.8 The closely related tricyclic 5[H]-
phenanthridin-6-ones (2) and aza-5[H]-phenanthridin-6-
ones (3a–c) are also well-known classes of inhibitors
whose structure–activity relationships against PARP-1
have recently been established.7,9 Preliminary findings
established that 4a10 (IC50=167 nM) was slightly more
potent against PARP-1 than 5[H]-phenanthridin-6-one
2, an unexpected result due to the planar nature of most
potent PARP-1 inhibitors (e.g., 1a–b, 2, and 3a–c).
Compound 4a also demonstrated increased intrinsic
solubility compared to 2 in common organic solvents.
This result coupled with previous findings indicating
enhanced in vitro and in vivo efficacy of 1-aza-5[H]-
phenanthridin-6-one 3a over 5[H]-phenanthridin-6-one
27 prompted the design and synthesis of partially satu-
rated derivatives of the aza-5[H]-phenanthridin-6-ones
(4b–d). In this manuscript, we present the synthesis and
in vitro activities of 1,3,4,5-tetrahydro-benzo[c][1,6]-
and [c][1,7]-napthyridin-6-ones as PARP-1 inhibitors
(4b–d, Fig. 1) whose core potencies are several times
better than the related unsaturated derivatives 3b–c.
Quinazolines (1a, Fig. 1) and isoquinolones (1b) are
examples of two ‘first generation’ PARP-1 inhibitors
The initial synthesis of these partially saturated aza-
5[H]-phenanthridin-6-ones is outlined in Scheme 1. The
first two steps are analogous to a literature synthesis of
*Corresponding author. Tel.: +1-410-631-8126; fax: +1-410-631-
6797; e-mail: ferrarisd@guilfordpharm.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00465-7

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D. Ferraris et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2513–2518
Figure 1. Structures of 5[H]-phenanthridin-6-ones and related PARP-1 inhibitors.
Scheme 1. (i) NaH/Tf2O/Et2O 95%; (ii) Pd(PPh3)4/K2CO3/DME, 8a=89%, 8b=97%; (iii) LiOH/THF/MeOH, 9a=83%, 9b=80%; (iv) (1)
DPPA/Toluene/Et3N, 85 ꢀC; (2) EtOH, 10a=66%, 10b=50%; (v) Eaton’s Reagent, 100 ꢀC, 11a=66%, 11b=33%.
a-1A adrenoreceptor agonists.11 Commercially available
ester 5 was converted to the triflate 6 using sodium
hydride and trifluoromethanesulfonic anhydride in 95%
yield. Suzuki coupling of 6 with either phenyl boronic
acid 7a or 4-fluorophenyl boronic acid 7b led to the
esters 8a and 8b in 89% and 97% yield, respectively.
The coupled products were hydrolyzed under basic
conditions to afford the carboxylic acids 9a (83%) and
9b (80%). The carboxylic acids were subsequently con-
verted into the ethyl carbamates 10a–b via a Curtius
rearrangement and quenching with ethanol. Cyclization
of the carbamates 10a–b was accomplished in moderate
yield (33–68%) by refluxing in Eaton’s reagent. The
final benzyl-protected 1,3,4,5-tetrahydro-benzo[c][1,6]-
napthyridin-6-ones 11a–b were formed in 10–39%
overall yield.
12 was accomplished in 75% yield to afford compound
13. The Suzuki coupling of 13 with phenyl boronic acid
was achieved in excellent yield to afford ester 14 in 87%
yield. The ester was subsequently hydrolyzed in high
yield (83%) to form carboxylic acid 15. The only
deviation from Scheme 1 occurs in the cyclization step.
The carboxylic acid 15 was converted to the isocyanate
in an analogous manner to acid 9a, but the
labile isocyanate was directly converted to the desired
N-benzyl-1,3,4,5-tetrahydro-benzo[c][1,7]-napthyridin-6-
one 16 in 25% yield by treatment with aluminum
trichloride.
Subsequent derivatization of 11a–b was carried out as
indicated in Scheme 3. Deprotection of benzyl amine
11a and 11b led to the secondary amines 4b and 4c after
workup in 93% yield. Acylation of the latent secondary
amine 4b and 4c with chloroacetyl-, propionyl- and
butyrylchlorides followed by amination led to the
tertiary amines 17a–b, 18a–b, 19a–b and 20.
The isomeric 1,3,4,5-tetrahydro-benzo[c][1,7]-napthyr-
idin-6-ones were prepared in a similar manner to 11a–b
as outlined in Scheme 2. Triflation of the starting ester

D. Ferraris et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2513–2518
2515
The glutamate derivative of 4b was also synthesized by
standard EDC coupling with Boc-Glu-OtBu to yield
amino acid 21 after deprotection. Sulfonamide deriva-
tives resulted from the addition of chloroethanesulfonyl
chloride to 4b in dichloromethane to afford the sulfonyl
olefin 22. Michael addition of piperidine and 4-pyrrolyl-
piperidine to 22 led to the tertiary amines 23a–b.
Similarly, deprotection of isomeric benzyl amine 16 in
.
MeOH/TFA led to 4d TFA as outlined in Scheme 4.
Scheme 2. (i) NaH/Tf2O/Et2O, 75%; (ii) Pd(PPh3)4/K2CO3/DME, 87%; (iii) LiOH/THF/MeOH, 83%; (iv) (1) DPPA/Toluene/Et3N, 85 ꢀC; (2)
AlCl3, 25%.
Scheme 3. (i) (1) H2/Pd/C/MeOH/TFA; (2) 10% K2CO3, 4b=93%, 4c=90%; (ii) (1) ClCO(CH2)nCl/Et3N, 71–85%; (2) R2NH (xs), 17a=55%,
17b=57%, 18a=59%, 18b=56%, 19a=44%, 19b=58%, 20=59%; (iii) (1) EDC/DMAP/DME, Boc-Glu-OtBu; (2) 10% TFA/DCM, 46% (2
steps); (iv) ClSO2CH2CH2Cl, Et3N, 48%; (v) R2NH, xs, 23a=58%, 23b=69%.

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D. Ferraris et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2513–2518
Scheme 4. (i) H2/Pd/C/MeOH/TFA, 78%; (ii) ClCO(CH2)nCl/Et3N, 51–67%; (2) R2NH (xs), 24a=47%, 24b=57%, 25a=62%, 25b=57%; (iii) (1)
EDC/DMAP/DCM/Et3N, Boc-Pro; (2) 10%TFA/DCM, 51% (2 steps) (iv) ClSO2CH2CH2Cl, Et3N, 53%; (v) R2NH, xs, 28a=59%, 28b=76%.
.
The secondary amine 4d TFA was acylated and ami-
nated in a similar manner to 4b and 4c leading to 24a–b,
Table 1. PARP-1 Inhibition of several tricyclic amides
Compd
Compound name
Structure
IC50 nMa
350
.
and 25a–b. Standard EDC coupling of 4d TFA with
Boc-proline, followed by deprotection of the Boc group
led to amine 26 in 53% yield. Sulfonamides 28a–b were
.
also synthesized from 4d TFA in a similar manner to
23a–b.
2
5[H]-Phenanthridin-6-one
4a
1,3,4,5-Tetrahydro-2H-
phenanthridin-6-oneb
167
The PARP-1 IC50’s of saturated and unsaturated 5[H]-
phenanthridin-6-ones and aza-5[H]-phenanthridin-6-
ones are outlined in Table 1.12 The parent tricyclic 5[H]-
phenanthridin-6-one 2 has an IC50 value of 350 nM
against PARP-1. By saturating two double bonds in the
C ring of 5[H]-phenanthridin-6-one, the potency increa-
ses by 2-fold as indicated by compound 4a (X, Y=CH2,
Fig. 1).10 Complete saturation of the C ring as shown by
1,3,4,4a,5,10b-Hexahydro-2H-phenanthridin-6-one,13
however, leads to a PARP-1 inhibitor that is over an
order of magnitude less potent than 5[H]-phenan-
thridin-6-one.
1,3,4,4a,5,10b-Hexahydro-2H-
phenanthridin-6-onec
7500
3b
4b
2-aza-5[H]-phenanthridin-6-one
128
35
1,3,4,5-Tetrahydro-2H-
benzo[c][1,6]naphthyridin-6-one
3c
3-aza-5[H]-phenanthridin-6-one
169
40
These data indicate that the A and B rings of these tri-
cyclic amides maintain potency when planar, a common
characteristic among several classes of PARP-1 inhibi-
tors.14 Substitution of a nitrogen for a carbon in the C
ring leads to the 2- and 3-aza-5[H]-phenanthridin-6-
ones (3b and 3c, Fig. 1), both of which exhibit activities
slightly better than the parent 5[H]-phenanthridin-6-
one. Likewise, saturation of the C ring of 3b and 3c
.
4d TFA
1,3,4,5-Tetrahydro-2H-
benzo[c][1,7]naphthyridin-6-one
aSee ref 12 for details.
bSee ref 10 for synthesis.
cSee ref 13 for synthesis.

D. Ferraris et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2513–2518
2517
4c and 19a–b maintained or increased the potency in
every instance as outlined in Table 2.
Table 2. PARP-1 Inhibition of several N-substituted 1,3,4,5-tetra-
hydro-2H-benzo[c][1,6] and [c][1,7]napthyridin-6-ones
The in vitro activities of N-substituted derivatives of 4d
are also outlined in Table 2. The parent compound
.
4d TFA is the most active member of the series
(IC50=30 nM) followed by the proline derivative 27a.
The amides 24a, 25a, have similar potencies to the pyr-
rolyl piperidine derivatives 24b and 25b. Slight loss in
potency (i.e., 2–5 times) was observed when changing
the amide bond to the sulfonamide linkage as illustrated
by compounds 28a–b.
Compd
R1
R2
n
IC50 nMa
EC50 nMb
4b
4c
H
F
H
H
H
35
20
30
59
42
25
457
162
240
185
99
40
40
45
60
58
30
4dTFA
17a
18a
19a
20
24a
25a
17b
18b
19b
24b
25b
23a
28a
H
H
F
1
2
2
3
1
2
1
2
2
1
2
In general, all of the 1,3,4,5-tetrahydro-benzo[c][1,6]-
[c][1,7]-naphthyridine analogues maintained a good
level of inhibition (<500 nM) regardless of the size or
hydrophobicity of the group. These results are indica-
tive of a large pocket adjacent to the nicotinamide
binding region of PARP-1, a result also noted in
previous publications.7,9
H
260
850
H
H
F
32
610
690
116
243
210
376
H
H
The in vitro cellular peroxide toxicity assay provided the
EC50 data outlined in Table 2.15 In general, the EC50 of
any given inhibitor was 1.5–3-fold less potent than the
corresponding IC50. The only exceptions were the
amino acid 21a and the sulfonamides 28a–b. These
results indicate that most of the 1,3,4,5-tetrahydro-ben-
zo[c][1,6]- and [c][1,7]-napthyridin-6-ones permeate the
cells in high enough concentration to inhibit PARP-1.
2790
23b
28b
357
349
1780
21a
27a
H
89
31
30,000
100
In conclusion, several potent PARP-1 inhibitors from
the 1,3,4,5-tetrahydro-benzo[c][1,6]- and [c][1,7]-nap-
thyridin-6-one class of compounds are presented in this
text. We have demonstrated the improvement in
potency upon partial saturation of the 5[H]-phenan-
aSee ref 12 for details.
bSee ref 15 for details.
.
leads to compounds 4b and 4d TFA (Fig. 1) whose
potencies are over three times better than their unsatu-
rated analogues. As a general trend, compounds with
partially saturated C rings are more potent than their
unsaturated cores in each case.
thridin-6-one and aza-5[H]-phenanthridin-6-one
C
rings. Finally, we have designed an efficient synthetic
route to several, different N-substituted 1,3,4,5-tetra-
hydro-benzo[c][1,6]- and [c][1,7]-napthyridin-6-ones as a
means to further improve the pharmacological proper-
ties of these inhibitors for potential use in acute,
ischemic injuries.
Several derivatives of compounds 4b are outlined in
Table 2. The parent compound, 4b, is among the most
potent members of this series (IC50=35 nM). The ter-
tiary amines 17a, 18a and 19a all displayed excellent
potencies against PARP-1 with the ethyl linker being
preferred (18a, 19a). A similar trend was noted for the
pyrrolylpiperazine moiety, that is, compounds 18b and
19b are slightly more potent than the one carbon ana-
logue 17b. Extension of the carbon chain to 3 methylene
units as in compound 20, however, decreased the activ-
ity several fold compared to the analogues with a
shorter linker. Changing from an amide to a sulfona-
mide linker also decreased the potency 3–4 times as
illustrated by compounds 23a–b. Glutamate derivative
21a, despite being several fold less potent than the core
4b, illustrates that hydrophobic substituents are not
necessary to maintain potency.
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Previous publications indicate that A-ring substituents
of 5[H]-phenanthridin-6-ones decrease inhibitory
potency,12 with the exception of a fluorine in the 8
position. As expected, the fluoro-substituted analogues

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PARP from Trevigan (Gaithersburg, MD, USA) was used to
determine the IC50 values of a PARP inhibitor. The PARP
enzyme assay is set up on ice in a volume of 100 mL consisting
of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl2, 30 mg/mL of
DNase activated herring sperm DNA (Sigma, MO, USA), 30
mM [3H]nicotinamide adenine dinucleotide (67 mCi/mmol), 75
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