Design and synthesis of poly ADP-ribose polymerase-1 inhibitors. 2. Biological evaluation of aza-5[H]-phenanthridin-6-ones as potent, aqueous-soluble compounds for the treatment of ischemic injuries

Article abstract of DOI:10.1021/jm030109s

A series of aza-5[H]-phenanthridin-6-ones were synthesized and evaluated as inhibitors of poly ADP-ribose polymerase-1 (PARP-1). Inhibitory potency of the unsubstituted aza-5[H]-phenanthridin-6-ones (i.e., benzonaphthyridones) was dependent on the positio

Full text of DOI:10.1021/jm030109s

3138
J . Med. Chem. 2003, 46, 3138-3151
Design a n d Syn th esis of P oly ADP -r ibose P olym er a se-1 In h ibitor s. 2. Biologica l
Eva lu a tion of Aza -5[H]-p h en a n th r id in -6-on es a s P oten t, Aqu eou s-Solu ble
Com p ou n d s for th e Tr ea tm en t of Isch em ic In ju r ies
Dana Ferraris,* Yao-Sen Ko, Thomas Pahutski, Rica Pargas Ficco, Larisa Serdyuk, Christina Alemu,
Chadwick Bradford, Tiffany Chiou, Randall Hoover, Shirley Huang, Susan Lautar, Shi Liang, Qian Lin,
May X.-C Lu, Maria Mooney, Lisa Morgan, Yongzhen Qian, Scott Tran, Lawrence R. Williams, Qi Yi Wu,
J ie Zhang, Yinong Zou, and Vincent Kalish
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, Maryland 21224
Received March 5, 2003
A series of aza-5[H]-phenanthridin-6-ones were synthesized and evaluated as inhibitors of poly
ADP-ribose polymerase-1 (PARP-1). Inhibitory potency of the unsubstituted aza-5[H]-phenan-
thridin-6-ones (i.e., benzonaphthyridones) was dependent on the position of the nitrogen atom
within the core structure. The A ring nitrogen analogues (7-, 8-, and 10-aza-5[H]-phenanthridin-
6-ones) were an order of magnitude less potent than C ring nitrogen analogues (1-, 2-, 3-, and
4-aza-5[H]-phenanthridin-6-ones). Preliminary stroke results from 1- and 2-aza-5[H]-phenan-
thridin-6-one prompted structure-activity relationships to be established for several 2- and
3-substituted 1-aza-5[H]-phenanthridin-6-ones. The 2-substituted 1-aza-5[H]-phenanthridin-
6-ones were designed to improve the solubility and pharmacokinetic profiles for this series of
PARP-1 inhibitors. Most importantly, three compounds from this series demonstrated statisti-
cally significant protective effects in rat models of stroke and heart ischemia.
In tr od u ction
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.1-3 The PARP-1 enzyme has
three structural regions: the DNA binding domain
containing two zinc fingers, the automodification do-
main, and the catalytic domain.4,5 The catalytic domain
is responsible for converting nicotinamide adenine di-
nucleotide (NAD+) into nicotinamide and an ADP-ribose
unit that is bound to the substrate protein or nucleotide
segment.6 When overactivated, PARP-1 can cause the
consumption of NAD+ and subsequently a drop in the
level of intracellular ATP. This depletion of ATP results
in cell death through a necrotic pathway and eventually
in ischemic tissue damage.7,8 Many studies with PARP-1
“knockout” mice have established the protective effects
against a stroke9 and a myocardial infarction (MI).10
More recently, studies have demonstrated the utility of
PARP-1 inhibitors in rat models of stroke and MI,11
indicating PARP-1 as a clinically relevant target for
ischemic injuries.
F igu r e 1. Structures of isoquinolones, 5[H]-phenanthridin-
6-ones and related PARP-1 inhibitors.
1. Accordingly, the lone pairs of the 5[H]-phenanthridin-
6-one oxygen bind to Ser904 and Gly863 while the
amide nitrogen donates a hydrogen bond to Gly863.15,16
Any manipulation of this amide portion of the ring
system results in decreased in vitro potency.17 There are
few examples, however, of PARP-1 inhibitors with
heteroatom replacements in the fused aryl rings of these
inhibitors (highlighted in bold, Figure 1).18 In this
manuscript, we present the aza-5[H]-phenanthridin-6-
ones (structure D; one X is N, and all other X groups
are CH) to better determine the in vitro and in vivo
effects of heteroatom replacements within the 5[H]-
phenanthridin-6-one core. We obtained information
about the inhibitory effects of a ring-nitrogen atom on
the nicotinamide binding pocket by testing the inhibi-
Many of the early PARP-1 inhibitors are based on the
structure of nicotinamide (Figure 1, structure A). For
example, quinazolines12 (Figure 1, structure B, X ) N),
isoquinolones13 (structure B, X ) CH), and the closely
related tricyclic 5[H]-phenanthridin-6-ones14 (structure
C, R ) H, Figure 1) are well-known classes of inhibitors
whose structure-activity relationships have been es-
tablished against PARP-1. It is generally accepted that
the 5[H]-phenanthridin-6-one core competes with NAD+
(structure A, Figure 1, R ) adenosine) for the nicoti-
namide binding pocket in the catalytic domain of PARP-
* To whom correspondence should be addressed. Phone: (410) 631-
8126. Fax: (410) 631-6797. Email: ferrarisd@guilfordpharm.com.
10.1021/jm030109s CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/11/2003

Aza-5[H]-phenanthridin-6-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3139
Sch em e 1a
a
Reagents: (i) Pd(PPh3)4, K2CO3, toluene/EtOH (10:1), 73-85%; (ii) THF, 3 equiv of LDA, 78-94%.
Sch em e 2a
tory potencies of 7-, 8-, and 10-aza-5[H]-phenanthridin-
6-ones against PARP-1. In addition, the 1-, 2-, 3-, and
4-aza derivatives were tested to determine the in vitro
and in vivo effects of a nitrogen atom in the C ring.
Structure-activity relationship data, as well as pre-
liminary rat stroke data, prompted us to derivatize this
1-aza-5[H]-phenanthridin-6-one core structure. Tertiary
amines were incorporated into this core as solubilizing
groups in an effort to administer these PARP-1 inhibi-
tors as intravenous solutions, a necessity for acute
clinical indications. Finally and most importantly, sev-
eral examples of PARP-1 inhibitors from the 2-substi-
tuted 1-aza-5[H]-phenanthridin-6-one series demon-
strate statistically significant efficacy in animal models
of brain and heart ischemia (MCAO, MI) by intravenous
administration.
Resu lts a n d Discu ssion
a
Reagents: (i) Pd(PPh3)4, 2.0 M Na2CO3, DME, 57%; (ii)
concentrated HCl, 60%.
Ch em istr y. The synthesis of 1-, 2-, and 4-aza-5[H]-
phenanthridin-6-ones 4a , 5, and 6 was carried out as
outlined in Scheme 1. Commercially available 2-chloro-
3-aminopyridine 2a was coupled with boronic acid 119
under standard Suzuki conditions to yield biphenyl 3a
in 85% yield. Subsequent cyclization occurred in THF
with the addition of 3 equiv of lithium diisopropylamide
(LDA) to afford the parent 1-aza-5[H]-phenanthrid-6-
one 4a in 89% yield. The 2- and 4-aza derivatives 520
and 6 were synthesized in a manner similar to that of
4a using 2b and 2c, respectively.
Sch em e 3a
The synthesis of 3-aza-5[H]-phenanthridin-6-one 9
required amide 7, which was synthesized according to
the literature procedure in 23% overall yield from
3-aminopyridine (Scheme 2).21 Coupling of amide 7 with
boronic acid 1 led to the amide 8 in 57% yield under
Suzuki coupling conditions. The treatment of amide 8
with concentrated HCl afforded 3-aza-5[H]-phenanthri-
din-6-one 9 in 60% yield. This material was identical to
the literature characterization of this compound.19
a
Reagents: (i) Pd(PPh3)4, K2CO3, toluene/EtOH (10:1), 29%; (ii)
H2SO4, NaNO2, 0-100 °C, 22%.
group have been reported.22 This one-step coupling
procedure was utilized with 14b, affording 15b in 19%
yield.
The 7-aza derivative was synthesized as indicated in
Scheme 3. The boronic acid carbamate 1122 was coupled
with 2-cyano-3-bromopyridine 10 under standard Su-
zuki conditions leading directly to amine 12 in 29%
yield. A diazotization of 12 was performed in H2O/H2-
SO4 with NaNO2 to afford the desired 7-aza-5[H]-
phenanthridin-6-one 13 in 22% yield.
The 2-substituted 1-aza-5[H]-phenanthridin-6-ones
were initially synthesized as illustrated in Scheme 5
(method A). Coupling of boronic acid 1 with com-
mercially available 2,6-dichloro-3-nitropyridine 16 led
to the nitro chloride 17a in 42% yield. The moderate
yield for this reaction can be explained by the isolation
of the isomeric 17b in approximately the same yield.
Chromatographic separation of the two isomers was
accomplished, and the desired isomer was aminated
with various amines to yield nitroamines 18b-g in
yields ranging from 72% to 92%. The subsequent reduc-
tion of the nitro group under standard hydrogenation
conditions led to the anilines 19b-g in excellent yields.
Synthesis of the 8- and 10-aza-derivatives 15a and
15b is outlined in Scheme 4. In one step, the coupling
of boronic acid 11 and 4-chloronicotinic acid ethyl ester
14a resulted in the cyclized 8-aza-5[H]-phenanthridin-
6-one 15a in one step in 24% yield. A similar cyclization
and subsequent deprotection of the butoxycarbonyl

3140 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Ferraris et al.
Sch em e 4a
by a combination of the last two steps of Scheme 5 into
one. Reduction of the nitro group with Raney nickel and
hydrazine, followed by immediate cyclization of the
amino ester, leads to 2-amino-1-aza-5[H]-phenanthridin-
6-one 4g in 29% overall yield compared to 14% overall
yield using method A. Amines 4h -m were also synthe-
sized via method B using the requisite amines in overall
yields of 20-30%. Salt formation was carried out as
illustrated in Scheme 5 to form (4g-m )‚HCl or (4g-
m )‚MsOH in 85-99% yield.
Further derivatization of amine 4d was carried out
as shown in Scheme 7. Acylation of 4d with chloroacetyl
chloride in dimethylacetamide led to the R-chloroamide
23 in 95% yield. Subsequent substitution of the acti-
vated chloride with various amines led to the subseries
of amides 24a -e in 82-92% yields.
Similarly, derivatization at the 3-position of 1-aza-
5[H]-phenanthridin-6-ones was accomplished by first
synthesizing 3-amino-1-aza-5[H]-phenanthridin-6-one
4n from boronic acid 1 and 2-chloro-3,5-dinitropyridine
25 using standard Suzuki coupling conditions (see
Scheme 7). Reduction of the intermediate dinitro ester
26 led to 4n . Reduction of the nitro groups led to an
intermediate diamine that cyclized to the desired
3-amino-1-aza-5[H]-phenanthridin-6-one 4n in 55% over-
all yield from 26. Acylation of this amine with chloro-
acetyl chloride led to the R-chloroamide 27 in 95% yield.
Chloride 27 was aminated in a manner similar to that
of the 2-substituted isomer 23 (Scheme 7) to afford
amides 28a -d .
Biologica l Eva lu a tion a n d SAR Discu ssion . A
comparative in vitro study of all aza-5[H]-phenanthri-
din-6-one derivatives with respect to the parent 5[H]-
phenanthridin-6-one (IC50 ) 350 nM, structure C,
Figure 1) is outlined in Table 1. Inhibitory potency of
these aza derivatives was found to be largely dependent
on which ring the nitrogen atom was incorporated.
While the A ring aza analogues exhibited significantly
lower potency than the parent 5[H]-phenanthridin-6-
one, all C ring aza analogues demonstrated similar or
slightly better potency. Inhibitory analysis of 13, 15a ,
and 15b demonstrates the deleterious effect of a nitro-
gen atom within the A ring of this core. The potencies
a
Reagents: (i) Pd(PPh3)4, K2CO3, toluene/EtOH (10:1), 19-24%.
The cyclization to 2-substituted 1-aza-5[H]-phenanthri-
din-6-ones was accomplished by using an excess of LDA
(3 equiv) in tetrahydrofuran. This cyclization procedure
afforded the desired compounds 4b-g in moderate
yields (42-46%). Salt formation was accomplished by
suspending the requisite free base in tetrahydrofuran
and addition of 1 equiv of acid (HCl in diethyl ether or
methanesulfonic acid). The hydrochloride or mesylate
salts (4b-g)‚HCl or (4b-g)‚MsOH precipitated out of
solution and were collected by filtration. The inherent
problems with method A occur with the LDA cyclization.
These strongly basic conditions limit the number of
functional groups that can be incorporated into the
synthesis. This limitation in addition to the low yields
led to the design of an alternative route (method B)
outlined in Scheme 6.
To directly compare the two methods, compound 4g
was prepared by method B. The boronic ester 20 was
prepared according to the literature23 on a gram scale.
The Suzuki coupling of 20 with 2,6-dichloro-3-nitropy-
ridine 16 led to the desired isomer 21a in 45% yield as
well as 35% of the undesired species 21b. Nucleophilic
aromatic substitution of chloride 21a was carried out
using N-methylpiperazine, leading to the nitro ester
22a . The improvement in the synthesis is accomplished
Sch em e 5a
a
Method A. Reagents: (i) Pd(PPh3)4, K2CO3, toluene/EtOH (10:1); (ii) HNR1R2 (excess), DIEA, 72-92%; (iii) H2/Pd/C, 90-100%; (iv)
THF, 3 equiv of LDA, 42-46%; (v) 1 equiv of HCl or MsOH, 84-95%.

Aza-5[H]-phenanthridin-6-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3141
Sch em e 6a
a
Method B. Reagents: (i) Pd(PPh3)4, K2CO3, toluene/EtOH (10:1); (ii) HNR1R2 (excess), DIEA, 77-90%; (iii) Raney nickel, H2NNH2,
36-95%; (iv) HCl or MsOH, 85-99%.
Sch em e 7a
a
Reagents: (i) Pd(PPh3)4, K2CO3, toluene/EtOH (10:1), 82%; (ii) (1) H2/Pd/C, MeOH, (2) THF, 3 equiv of LDA, 55% overall; (iii) chloroacetyl
chloride, Et3N, DMA, 95%; (iv) HNR2 (excess), DMA, 82-92%.
Ta ble 1. PARP-1 Inhibition of Aza-5[H]-phenanthridin-6-ones
pocket of the catalytic domain. If this holds true, the
b
potency of 15a can be explained by an analogous
difference in potency between benzamide (IC50 ) 20 µM)
and nicotinamide (IC50 ) 210 µM).24 The decrease in
potency for 13 and 15b may be attributable to the ring
nitrogen interacting electrostatically with residues in
the hydrophobic nicotinamide binding pocket (His862,
Gly863, Phe897, or Ala898). Compound 13 is the least
active of these three presumably because the ring
nitrogen not only interacts electrostatically with the
hydrophobic binding pocket but adversely affects the
hydrogen bond formed by Ser904 on the amide carbonyl.
The inhibition results from the (1-4)-aza derivatives
4a , 5, 6, and 9 also confirm previous findings about 5[H]-
phenanthridin-6-one SAR,14 namely, the tolerance for
hydrophilic moieties on the C ring.
compound
name
IC50 (nM)
benzamidea
20000
nicotinamidea
210000
350 ( 8.9c
116 ( 2.9c
128 ( 3.9c
169
C (R ) H)
4a
5
9
6
13
15a
15b
5[H]-phenanthridin-6-one
1-aza-5[H]-phenanthridin-6-one
2-aza-5[H]-phenanthridin-6-one
3-aza-5[H]-phenanthridin-6-one
4-aza-5[H]-phenanthridin-6-one
7-aza-5[H]-phenanthridin-6-one
8-aza-5[H]-phenanthridin-6-one
10-aza-5[H]-phenanthridin-6-one
311
14000
2600
2900
See ref 24. b See Experimental Section for details. c Represents
a
an average of three IC50 values.
of compounds 13 (IC50 ) 14 µM), 15a (IC50 ) 2.6 µM),
and 15b (IC50 ) 2.9 µM) were up to an order of
magnitude worse than 5[H]-phenanthridin-6-one, while
C ring derivatives 4a , 5, 6, and 9 all demonstrated
similar or slightly better potency than 5[H]-phenan-
thridin-6-one. Presumably, the A ring of these aza-5[H]-
phenanthridin-6-ones binds to the nicotinamide binding
While the enzymatic potencies of 4a and 5 were
slightly better than the potency of 5[H]-phenanthridin-
6-one, preliminary stroke data on 4a and 5 displayed
significant protective effects versus 5[H]-phenanthridin-

3142 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Ferraris et al.
Ta ble 2. PARP-1 Inhibition of 2-Substituted
1-Aza-5[H]-phenanthridin-6-ones 4b-m
Ta ble 3. PARP-1 Inhibition of
1-Aza-5[H]-phenanthridin-6-ones 24a -d and 28a -d
a
See Experimental Section for details. n.d. ) not determined.
with the desired PARP-1 inhibitor and consequently
damaged with H2O2. The concentration of the inhibitor
required to achieve 50% reduction in cell death is
reported as EC50. For the 2-amino-1-aza-5[H]-phenan-
thridin-6-ones 4e-g and 4j‚MsOH, the cellular activi-
ties (EC50) were approximately 2-5 less potent com-
pared to the corresponding enzymatic IC50 values. The
one exception to this trend was N-oxide 4l whose EC50
was over 20 times less potent than the inhibitory
constant. This result indicates that this compound,
despite being a metabolite for 4g, does not adequately
penetrate into cells and is most likely not responsible
for in vivo efficacy of 4g in animal models of brain
ischemia as discussed below.
The in vitro potencies of the acyl 2- and 3-substituted
derivatives of 4a are listed in Table 3. Most of these
derivatives were potent against PARP-1 regardless of
the size of the substituent. The bulkiest derivative 24d
(IC50 ) 277 nM) was only 2-3 times less potent than
the most active derivatives 24a and 28d . Once again,
the steric bulk of these derivatives underscores the
versatility of the 2- and 3-positions to modulate the
pharmaceutic and pharmacokinetic profiles of this
series.
a
b
Average of three IC50 values. Made from deprotection of 4h .
c Methylation product of 4i. Oxidation product of 4g; see Scheme
d
8. e See Experimental Section for details. n.d. ) not determined.
6-one (vide infra). These data prompted further deriva-
tization of the 1-aza and/or 2-aza analogues. On the
basis of previous 5[H]-phenanthridin-6-one SAR data,14
the 2- and 3-positions are best suited for substitution
while maintaining inhibitory potency. Because of this
precedent, derivatives of the 1-aza-5[H]-phenanthridin-
6-one core 4a were pursued, since both the 2- and
3-positions can be substituted while derivatives of 5 can
only be substituted from the 3-position.
The 2-substituted derivatives 4b-m were assayed
against PARP-1, and their potencies are outlined in
Table 2. The ethylenediamino derivatives 4b and 4c had
activities that were slightly higher than the parent 4a
(IC50 ) 116 nM, Table 1). A slight improvement in IC50
occurred with the addition of a piperazine moiety, as
seen for compounds 4g, 4j‚MsOH, and 4m ‚MsOH. In
fact, this activity was even maintained for the bicyclic
derivatives 4i‚TFA and 4k ‚MsOH. Compound 4h , the
The cellular activities for the amides 24a -d and
28a -d , however, were notably less potent than their
respective IC50 values. Compound 24b had the most
potent EC50 value within the series (1110 nM), a 7-fold
decrease from its IC50. In addition, there is an order of
magnitude difference in cellular activity between the
2- and 3-amido-1-aza-5[H]-phenanthridin-6-ones and
the corresponding 2-substituted piperazines outlined in
Table 2.
precursor to analogues 4i‚TFA and 4k ‚MsOH (IC50
)
774 nM), was the least active analogue, perhaps because
of the size of the butoxycarbonyl group and/or the
necessity for a charged amine functionality. The hydro-
philic N-oxide 4l (IC50 ) 247 nM), a major metabolite
of 4g as discussed vide infra, was 5-6 times less potent
than its unoxidized precursor 4g (IC50 ) 45 nM).
An H2O2 cytotoxicity assay was utilized to determine
the effectivness of PARP-1 inhibitors in penetrating cells
and preventing cell death.25 The cells were preincubated
P h a r m a cology Da ta
Solu bility Da ta . Because of the structural charac-
teristics of most PARP-1 inhibitors (i.e., highly conju-
gated, crystalline, and planar), poor aqueous solubility
is a significant issue. To develop a drug for acute
ischemic injuries (i.e., stroke, MI), the compound should
ideally be soluble in intravenous buffers (e.g., citrate,

Aza-5[H]-phenanthridin-6-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3143
F igu r e 2. Pharmacokinetic profile of 4g‚MsOH.
F igu r e 3. Pharmacokinetic profile of 24c‚HCl.
Ta ble 4. Solubility Measurementsa
turbidity
turbidity
concn
measurement measurement
compound (mg/mL)
salt
HCl
MsOH
MsOH
MsOH
MsOH
MsOH
HCl
HCl
HCl
HCl
HCl
(CBb)
(PBSc)
4g
4g
5
5
13.0
1.3
4.0
45.7
17
11.5
143
12.7
13.0
21.0
25.3
26.8
1.4
145
215
pcp
4g
4g
4e
20
40
5
4m
24a
24c
24c
24c
24d
5
5
5
20
40
5
pcp
190
17.4
15.6
pcp
63.1
a
All measurements were taken on
a
Nepheloskan Ascent
instrument, type 750, manufactured by Labsystems. Baseline
reading ) 0.1. Turbidity measurement greater than 20 indicates
b
that the compound is insoluble. pcp ) precipitate. CB ) 50 mM
citrate buffer, pH ) 4.0. c PBS ) 70 mM phosphate-buffered saline
solution, pH ) 7.4.
Ta ble 5. Cmax Values for Selected PARP-1 Inhibitors in Rats
(10 mg/kg, n ) 6)a
Cmax
compound
plasma (µg/mL)
heart (µg/g)
brain (µg/g)
4g‚MsOH
4m ‚MsOH
24c‚HCl
4l
0.456
1.95
4.79
0.010
3.79
2.98
7.48
38.4
nd
13.7
8.85
1.44
nd
4j‚MsOH
20.3
2.18
a
Compounds were injected intravenously using 70 mM PBS as
1-aza-5[H]-phenanthridin-6-ones (administered intra-
venously in citrate buffer). A notable feature of these
data is the structural similarity between piperazines
4g‚MsOH and 4m ‚MsOH and the requisite similarities
in distribution. These two compounds have the highest
Cmax values of those tested, especially in the brain. A
graphical representation of the PK profile of 4g‚MsOH
is illustrated in Figure 2. There is, however, a noticeable
PK profile difference between these piperazines and
amide derivative 24c‚HCl (Figure 3). Namely, amide
24c‚HCl does not achieve brain levels as high as the
piperazines, yet 24c‚HCl does have a much higher heart
level and a much longer half-life (consequently a larger
area under the curve (AUC)), as seen in the graphical
depiction in Figure 3. All three compounds (4g‚MsOH,
4m ‚MsOH, and 24c‚HCl) have higher concentrations in
the heart and brain than the plasma (4-30 times
higher). The ability to achieve high tissue concentrations
emphasizes the permeability of these PARP-1 inhibitors
and could be beneficial for treating stroke and heart
ischemia, given that the enzyme target (PARP-1) is
tissue-based.
The PK profile for 4g‚MsOH (Figure 2) illustrates a
relatively short half-life but relatively high Cmax in
tissues. This short half-life indicates that the parent
compound is rapidly metabolized in the rat, causing
depletion of the parent compound. To address the
metabolism issue, 4g‚MsOH was subjected to liver
microsomal enzymes and the byproducts were analyzed
by LC/MS. The major metabolites result from the
oxidative degradation of the piperazine ring. The major
product resulting from the microsomal exposure of
4g‚MsOH had a mass of 310, which corresponds to the
parent compound plus one oxygen atom. Because there
are multiple potential sites of oxidation, we conducted
a synthetic effort to establish the most prevalent
the buffer. nd ) not detected.
phosphate) and deliverable by bolus injection or in-
fusion. To determine the relative solubility of many of
these analogues, turbidity measurements were taken
at 5-20 mg/mL in two common buffers, namely, citrate
buffer (CB) and phosphate buffered saline (PBS) (Table
4).26 These solubility data acted as a guideline in
determining the derivatives that could be tested as
intravenous solutions in vivo and the maximum con-
centrations at which they could be administered. Con-
centrations that had turbidity measurements higher
than 20 were considered insoluble and not used for
administration in animal models.
In general, most piperazine mesylate salts (4e‚MsOH,
4g‚MsOH, and 4m ‚MsOH) were soluble in the 50 mM
citrate buffer at 5 mg/mL. Piperazine 4g, one of the most
soluble compounds within the series, was soluble as both
the HCl and MsOH salt in CB, even though the HCl
salt only had limited solubility in PBS. Compound
4g‚MsOH was the only piperazine with a high concen-
tration limit of 20 mg/mL in citrate buffer. Of all the 2-
and 3-amido derivatives tested, only compound 24c‚HCl
was soluble in any buffer by turbidity measurements.
The high concentration limit for amide 24c‚HCl was 20
mg/mL in CB. These parameters were eventually used
as a guideline for in vivo administration in the models
discussed below.
In Vivo Resu lts. Dr u g Meta bolism a n d P h a r m a -
cok in etic Da ta . After the aqueous solubility for the
amine and amide derivatives of 4a was established, the
next logical step was to obtain drug metabolism and rat
pharmacokinetic (PK) data for representative com-
pounds from each subseries to prioritize the compounds
for in vivo testing. Table 5 outlines the Cmax values in
the plasma, heart, and brain for selected 2-substituted

3144 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Ferraris et al.
Sch em e 8a
a
Reagents: (i) mCPBA, DCE, 75%, or rat microsomal enzymes.
F igu r e 4. Metabolism of compound 4g (Wistar rat).
Ta ble 6. Cerebral Ischemic Stroke Results
dose
reduction in
infarct volume (%) MCAO model
F oca l Cer ebr a l Isch em ic Str ok e Da ta . On the
basis of their solubility and pharmacokinetic profiles,
compounds 4g‚MsOH and 24c‚HCl were chosen for
animal brain ischemia studies. A widely accepted ani-
mal model of brain ischemia is the transient MCAO
stroke model.31 Table 6 outlines the effects of the
PARP-1 inhibitors that were tested in this model. These
compounds were administered 30 min before occlusion
of the middle cerebral artery and immediately after
reperfusion in a bolus dose (20 or 40 mg/mL iv in CB or
ip in DMSO over 3 min for 4a , 5, and C). After several
hours, the stroked animals were sacrificed and brain
slices were analyzed to calculate the overall area of
infarction or dead tissue. Reduction in this area repre-
sents a protective effect and is noted in Table 6 as a
reduction in infarct volume. The initial results for this
model were obtained with the parent aza-5[H]-phenan-
thridin-6-ones 4a and 5 via intraperitoneal administra-
tion. Compound 4a showed a strong trend toward
reduction in infarct volume when dosed at 40 mg/kg 30
min before ischemia and 40 mg/kg after reperfusion.
This type of effect was mirrored by the 2-aza analogue
5. These initial results were surprising because 5[H]-
phenanthridin-6-one did not show protection in the same
model. Clearly, the only limits to persuing 4a and 5 as
potential drug candidates are their innate aqueous
solubility and inability to be administered intra-
venously. For this reason, the amine salts 4g‚MsOH and
24c‚HCl were designed and synthesized to overcome
these shortcomings. The results for 4g‚MsOH were very
encouraging, as illustrated in Table 6. At the 40 mg/kg
total dose, 4g‚MsOH resulted in a 44% reduction in
infarct volume in this transient model. The majority of
this protective effect was even maintained at the lower
dose of 20 mg/kg. The two potential metabolites of 4g,
namely, 4j and 4l, are both viable inhibitors of PARP-1
and could potentially account for some of the efficacy
in this MCAO model (Table 2). The pharmacokinetic
analysis, however, indicates that the N-oxide 4l does
not penetrate readily into tissues and hence is most
likely not responsible for in vivo efficacy in this model.
In addition, the minimal amount of 4j available from
metabolism (2 orders of magnitude less than 4g) indi-
cates that 4j is probably not responsible for the efficacy
either.
a
compound (mg/kg)
n
4a
5
80b
40b
40b
40c
20c
80c
80c
40c
10
10
10
10
10
10
10
10
55 (F ) 0.055)
47 (F ) 0.031)
-7 (F ) nsf)
44 (F ) 0.008)
33 (F ) 0.017)
-1 (F ) nsf)
35 (F ) 0.039)
18 (F ) 0.02)
transientd
transientd
transientd
transientd
transientd
transientd
permanente
permanente
C (R ) H)
4g‚MsOH
4g‚MsOH
24c‚HCl
4g‚MsOH
4g‚MsOH
a
b
n ) number of rats per experiment. Compound was admin-
istered intraperitoneally. c Compound was administered intrave-
d
nously. Half of compound was administered as a bolus dose 30
min before ischemia, and half was administered 30 min after
reperfusion. See ref 31. e Half of compound was administered as
a bolus dose 30 min before ischemia, and half was administered
30 min after ischemia. See ref 32. f ns ) not significant.
metabolite. When compound 4g was subjected to 1 equiv
of m-CPBA, the major product was compound 4l,27 one
potential metabolite (Scheme 8). This similar oxidation
appeared to be rapidly catalyzed by either a flavin
monooxygenase28 or a cytochrome P450 enzyme29 within
the microsomal culture. Direct comparison of the mi-
crosomal culture with a pure sample of 4l confirmed this
as the major metabolite. Compound 4l displayed a
drastically different PK profile than the parent 4g, as
outlined in Table 5. This material 4l, due to the presence
of a polar N-oxide moiety, was not detected in the brain
or heart tissue.
Another minor product from the microsomal degrada-
tion study displayed a mass of 279, corresponding to the
parent compound without a methyl group. The des-
methyl compound 4j was synthesized as outlined in
Scheme 6 and directly compared to the microsomal
samples. Compound 4j‚MsOH (IC50 ) 58 nM, Table 3),
whose free base is a product resulting from oxidative
demethylation30 of the piperazine methyl group, dis-
played a different PK profile than the metabolic precur-
sor 4g‚MsOH (Table 6). That is, the Cmax for 4j was
6-fold lower in the brain while the Cmax for 4j was almost
10-fold higher in the heart than 4g‚MsOH. A graphical
representation of the metabolism of 4g‚MsOH in Wistar
rats is illustrated in Figure 4 (plasma only). The
appearance of 4l and, to a lesser extent, the desmethyl
compound 4j proceeds immediately, presumably because
of rapid oxidation or demethylation of 4g. The rapid
clearance of the piperizine 4g happens while the two
major metabolites 4j and 4l are detected. Once formed,
4j and 4l disappear at a rate similar to that of 4g.
Unfortunately, amide 24c‚HCl did not afford the same
level of protection as 4g‚MsOH. In fact, at an 80 mg/kg
total dose, 24c‚HCl did not show any reduction in infarct
volume. One possible explanation for this result can be

Aza-5[H]-phenanthridin-6-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3145
Ta ble 7. Rat Myocardial Ischemia Data
Con clu sion s
doseb
reduction in
We have designed and synthesized a series of aza-
5[H]-phenanthridin-6-ones as potent PARP-1 inhibitors
with clearly defined structure-activity relationships.
These SAR studies further elucidate the hydrophobic
constraints of the nicotinamide binding pocket by
demonstrating the lack of activity of aza-5[H]-phenan-
thridin-6-ones 13, 15a , and 15b. The aza-5[H]-phenan-
thridin-6-one core has shown in vitro activity similar
to that of 5[H]-phenanthridin-6-one but has shown a
notably better in vivo efficacy in ischemic models as
displayed by compounds 4a and 5. Derivitization of the
1-aza-5[H]-phenanthridin-6-one core led to several ex-
amples of potent PARP-1 inhibitors (42-100 nM). In
addition to the enzymatic activity, this series contains
examples of compounds with ionizable groups for in-
creased aqueous solubility and thus potential clinical
utility in acute settings. We established Cmax values for
selected inhibitors to predict in vivo efficacy in animal
models of ischemia. Most importantly, we have demon-
strated three 1-aza-5[H]-phenanthridin-6-one PARP-1
inhibitors (4g, 4m , and 24c) that are protective when
administered intravenously in animal models of heart
ischemia and stroke.
a
compound
vehicle
n
(mg/kg) infarct volume (%)
4g‚MsOH
4g‚MsOH
4m ‚MsOH
24c‚HCl
24c‚HCl
24c‚HCl
70 mM PBS
70 mM PBS
50 mM CB
50 mM CB
50 mM CB
50 mM CB
8
80
40
40
80
40
20
25.5 (F ) 0.013)
18.8 (F ) 0.08)
36 (F ) 0.001)
44 (F ) 0.001)
39 (F ) 0.007)
13 (F ) 0.15)
8
8
8
8
8
a
b
n ) number of rats per experiment. Half of compound was
administered intravenously 5 min before ischemia and 5 min
before reperfusion.
gathered from the pharmacokinetic data for these two
compounds. Analogue 4g‚MsOH, while having a sig-
nificantly shorter half-life than 24c‚HCl, does have a
higher Cmax in the brain. Perhaps this high initial
concentration of drug is necessary to achieve a protec-
tive effect in ischemic models such as the rat transient
MCAO. This result also illustrates that Cmax may be a
more relevant PK parameter than the half-life or AUC
for predicting the protective effects of PARP-1 inhibitors
in brain ischemia. That is, compound 24c‚HCl has a
longer half-life (and larger AUC) in the brain than
4g‚MsOH but its Cmax is 10 times lower than that of
4g‚MsOH.
Exp er im en ta l Section
A more stringent animal model of brain ischemia is
the rat permanent MCAO model.32 In this model, the
middle cerebral artery is permanently occluded as
opposed to the temporary occlusion of the transient
model. Infarct volume was determined in a manner
similar to the transient model, and this value is recorded
in Table 6 as percent reduction in infarct volume. The
PARP-1 inhibitor 4g‚MsOH was administered 30 min
before ischemia at 40 mg/kg and 30 min after ischemia
at 40 mg/kg and still demonstrated a significant reduc-
tion in infarct volume (35%). In fact, lowering the dose
to 20 mg/kg before and 20 mg/kg after also resulted in
an 18% reduction.
Gen er a l. Melting points were obtained on a MEL-TEMP
II and were uncorrected (Meltemp Laboratory Devices, Inc.).
Proton nuclear magnetic resonance were recorded at 400 MHz
on a Bruker 400 using deuterated solvent as an internal
standard. Chemical shift values are indicated in parts per
million. Mass spectra were recorded using a Micromass LCS
Platform LC/MS spectrometer. Elemental analyses were ob-
tained from Atlantic Microlabs, Inc. (Norcross, GA). All
reagents were purchased from Aldrich Chemical (Milwaukee,
WI) or CB Reasearch (New Castle, DE) unless otherwise
stated.
Gen er a l P r oced u r e for th e Syn th esis of 2-(3-Am in op y-
r id in -2-yl)-N,N-d iisop r op ylben za m id e (3a ). The boronic
acid 1 (2.0 g, 8.0 mmol), prepared according to the literature,19
was added to a solution of potassium carbonate (2.2 g in 8 mL
of H2O) and 2-chloro-3-amino pyridine 2a (0.94 g, 7.3 mmol)
in 100 mL of toluene/EtOH (9:1). This mixture was deoxygen-
ated in vacuo and refilled with nitrogen. After the mixture was
stirred under nitrogen for 30 min, palladium tetrakistri-
phenylphosphine (420 mg, 0.36 mmol) was added to the
mixture. The solution was heated to 80 °C until complete
conversion was attained according to TLC (50:50 hexanes/
EtOAc). The solvent was removed in vacuo, and the reaction
mixture was then partitioned between water (100 mL) and
EtOAc (100 mL). The water layer was extracted two more
times with EtOAc (100 mL), and the combined organic layers
were dried with Na2SO4 and concentrated to yield a crude solid
that was triturated with diethyl ether (10-20 mL) to yield the
desired amine 3a as a yellow solid (1.84 g, 85%): 1H NMR
(CDCl3) δ 8.05 (d, 1H), 7.45 (m, 3H), 7.28 (d, 1H), 7.06 (m,
1H), 7.00 (d, 1H), 4.01 (s, 2H), 3.78 (m, 1H), 3.31 (m, 1H), 1.48
(d, 3H), 1.13 (d, 3H), 1.01 (d, 3H), 0.84 (d, 3H); MS (ES+) )
298. Anal. (C18H23N3O) C, H, N.
Myoca r d ia l Isch em ia Da ta . If Cmax is also the
relevant pharmacokinetic parameter for heart ischemia,
one would predict that the in vivo efficacy of 24c‚HCl
would be better in the rat model of myocardial infarction
than 4g‚MsOH.33,34 While the brain levels for compound
4g‚MsOH are higher than those for 24c‚HCl, their
relative concentrations in the heart are juxtaposed. That
is, 24c‚HCl has maximum heart levels that are 13 times
higher than 4g‚MsOH (Table 5). The protective results
from these two compounds are outlined in Table 7. As
expected, compound 4g‚MsOH displayed a modest
reduction in infarct volume at the two doses tested (40
mg/kg 5′ before ischemia, 40 mg/kg 5′ before reperfusion)
while amide 24c‚HCl, at this same dose (80 mg/kg),
reduced the infarct volume by 44%. Even at the 40 mg/
kg total dose, 24c‚HCl maintained efficacy (39%).
Similarly, compound 4m ‚MsOH displayed better protec-
tion at the 40 mg/kg dose than 4g‚MsOH, correlating
with their relative Cmax values in the heart tissue. These
in vivo results once again illustrate the utility of Cmax
as a pharmacokinetic parameter to predict efficacy in
an ischemic model. All together, these data indicate that
PARP-1 inhibitors can potentially be used to reduce the
damage caused by myocardial infarction.
2-(4-Am in opyr idin -3-yl)-N,N-diisopr opylben zam ide (3b).
4-Amino-3-iodopyridine 2b (1.4 g, 8.1 mmol), boronic acid 1
(2.0 g, 8.9 mmol), and potassium carbonate (2.2 g, 15.9 mmol)
were dissolved in 80 mL of toluene, 8 mL of EtOH, and 8 mL
of H2O. This mixture was purged of oxygen and refilled with
nitrogen several times. Then, tetrakistriphenylphosphine-
palladium (350 mg, 0.30 mmol) was added to the mixture, and
the mixture was heated to 80 °C overnight. Water (100 mL)
was then added to the reaction mixture, and the organic layer
was partitioned. The aqueous layer was extracted with EtOAc
(2 × 100 mL), and the combined organics were dried with Na2-

3146 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Ferraris et al.
SO4 and concentrated. The crude reaction product was tritu-
rated with diethyl ether (25 mL) and filtered. The resulting
solid was collected and characterized as the biphenylamine
3b (2.0 g, 83%): 1H NMR (CDCl3) δ 8.17 (d, 1H), 8.03 (s, 1H),
7.45 (m, 2H), 7.25 (m, 2H), 6.55 (d, 1H), 4.50 (bs, 2H), 3.61
(m, 1H), 3.31 (m, 1H), 1.49 (d, 3H), 1.16 (d, 3H), 1.03 (d, 3H),
0.83 (d, 3H); MS (ES+) ) 298. Anal. (C18H23N3O) C, H, N.
2-(2-Am in opyr idin -3-yl)-N,N-diisopr opylben zam ide (3c).
The synthesis was carried out in a manner identical to that of
3b (73%): 1H NMR (CDCl3) δ 7.94 (d, 1H), 7.44 (m, 2H), 7.29
(m, 2H), 6.58 (m, 1H), 5.49 (bs, 2H), 3.56 (m, 2H), 1.40 (d, 3H),
1.05 (d, 3H), 0.93 (d, 3H), 0.64 (d, 3H). Anal. (C18H23N3O) C,
H, N.
mmol), potassium carbonate (1 g, 7.25 mmol), and tetraki-
striphenylphosphinepalladium (100 mg, catalyst) were mixed
together in toluene (50 mL) and ethanol (5 mL) and heated to
70 °C overnight. The reaction was quenched with water (75
mL), and the mixture was then extracted with EtOAc (3 × 50
mL). The combined organics were dried and concentrated in
vacuo and chromatographed (50:50 EtOAc/hexanes to 10%
MeOH/EtOAc) to yield the amine 12 (235 mg, 29%): mp )
294-298 °C; 1H NMR (DMSO-d6) δ 8.86 (d, 1H), 8.75 (d, 1H),
8.28 (d, 1H), 7.72 (m, 2H), 7.61 (t, 1H), 7.38 (t, 1H), 6.21 (bs,
2H); MS (ES+) ) 196. Anal. (C12H9N3) C, H, N.
7-Aza -5[H]-p h en a n th r id in -6-on e (13). The aniline 12 (50
mg, 0.25 mmol) was suspended in 1 mL of H2O and 0.7 mL of
H2SO4 and cooled to 0 °C. A solution of NaNO2 (18 mg, 0.26
mmol in 0.2 mL of H2O) was added to the acidic solution
dropwise. The reaction mixture was warmed to room temper-
ature and heated to 100 °C for 3 h. The mixture was then
cooled to room temperature, and some particulates were
filtered off. The filtrate was set out overnight, and the resulting
crystals were filtered off and triturated with boiling EtOAc
and filtered. The resulting solid was characterized as the
desired material 13 (11.2 mg, 22%): mp ) 300-310 °C; 1H
NMR (DMSO-d6) δ 11.91 (s, 1H), 8.98 (d, 1H), 8.43 (d, 1H),
7.85 (m, 1H), 7.55 (m, 2H), 7.38 (m, 2H); MS (ES+) ) 197. Anal.
(C12H8N2O) C, H, N.
1-Aza -5[H]-p h en a n th r id in -6-on e (4a ). The amine 3a
(1.74 g, 5.8 mmol) was dissolved in dry tetrahydrofuran (25
mL), and the mixture was cooled to -78 °C under nitrogen.
Lithium diisopropylamide (2.0 M, 7.6 mL) was added dropwise
to the solution, and this mixture was stirred for several hours
and warmed to room temperature overnight. The reaction was
quenched with water (50 mL), and the mixture was extracted
with 10% MeOH/CH2Cl2. The combined organics were dried
and concentrated to yield the crude solid, which was triturated
with boiling diethyl ether to yield the pure material 4a as a
1
yellow solid (0.95 g, 89%): mp ) 300-320 °C (dec); H NMR
(DMSO-d6) δ 11.78 (s, 1H), 8.77 (d, 1H), 8.55 (d, 1H), 8.32 (d,
1H), 7.93 (d, 1H), 7.74 (m, 2H), 7.54 (m, 1H); MS (ES+) ) 197.
Anal. (C12H8N2O) C, H, N.
8-Aza -5[H]-p h en a n th r id in -6-on e (15a ). 4-Chloronicotinic
acid ethyl ester 14a (500 mg, 2.7 mmol), boronic acid 10 (1.5
g, 6.1 mmol), sodium carbonate (715 mg, 6.6 mmol), and
tetrakis triphenylphosphinepalladium (140 mg, catalyst) were
suspended in DME (30 mL) and heated to 80 °C overnight.
The solvent was removed in vacuo and the residue was
chromatographed on silica gel (50% EtOAc/hexanes to 10%
MeOH/EtOAc) to yield the desired compound 15a as a yellow
2-Aza-5[H]-ph en an th r idin -6-on e (5). A solution of lithium
diisopropylamide (2.0 M, 10 mL) was dissolved in 90 mL of
THF, and the mixture was cooled to -78 °C. A solution of
amine 3b (2.0 g, 6.73 mmol) in THF (25 mL) was added to the
LDA dropwise over a 15 min period. The reaction mixture was
warmed to room temperature and stirred overnight. The
reaction mixture was concentrated in vacuo and suspended
in 100 mL of water. The solid was filtered off and triturated
with ethyl acetate (100 mL). The resulting solid was dried to
yield the desired compound 5 (1.24 g, 94%): mp ) 300-320
1
solid (124 mg, 24%): mp ) 295-300 °C; H NMR (DMSO-d6)
δ 11.80 (bs, 1H), 9.43 (s, 1H), 8.91 (d, 1H), 8.43 (m, 2H), 7.62
(t, 1H), 7.32 (m, 2H); MS (ES+) ) 197. Anal. (C12H8N2O) C, H,
N.
1
°C (dec); H NMR (DMSO-d6) δ 11.57 (bs, 1H), 9.57 (s, 1H),
10-Aza -5[H]-p h en a n th r id in -6-on e (15b). The same pro-
cedure was used as 15a with boronic ester 10 and 2-chloro-
picolinic acid ethyl ester 14b to afford 15b as a yellow solid
(34%): mp ) 295-300 °C; 1H NMR (DMSO-d6) δ 11.90 (bs,
1H), 9.06 (d, 1H), 8.63 (m, 2H), 7.69 (m, 1H), 7.60 (t, 1H), 7.40
(d, 1H), 7.32 (m, 1H); MS (ES+) ) 197. Anal. (C12H8N2O) C,
H, N.
Meth od A. Syn th esis of 2-(6-Ch lor o-3-n itr op yr id in -2-
yl)-N,N-d iisop r op ylben za m id e (17a ). The boronic acid 1
(2.0 g, 8.0 mmol) was added to a solution of potassium
carbonate (2.2 g in 8 mL of H2O) and 2,5-dichloro-3-nitropy-
ridine 16 (1.4 g, 7.3 mmol) in 100 mL of toluene/EtOH (8:1).
This mixture was deoxygenated in vacuo and refilled with
nitrogen. After the mixture was stirred under nitrogen for 30
min, tetrakistriphenylphosphinepalladium (250 mg) was added
to the mixture. The solution was heated to 80 °C until complete
conversion (no starting material) according to TLC (50:50
hexanes/EtOAc). The reaction mixture was then extracted with
water and the toluene layer was dried and concentrated to
yield a crude oil that was columned on silica gel to afford the
desired isomer 17a as a yellow oil (1.20 g, 42%): 1H NMR
(CDCl3) δ 8.26 (d, 1H), 7.45 (m, 3H), 7.34 (m, 2H), 3.99 (m,
1H), 3.41 (m, 1H), 1.38 (bs, 9H), 1.21 (d, 3H); MS (ES+) ) 363.
Anal. (C18H20ClN3O3) C, H, N.
8.65 (d, 1H), 8.50 (d, 1H), 8.33 (d, 1H), 7.90 (t, 1H), 7.71 (t,
1H), 7.28 (d, 1H); MS (ES+) ) 197. Anal. (C12H8N2O) C, H, N.
4-Aza -5[H]-p h en a n th r id in -6-on e (6). The cyclization was
done in a manner similar to the cyclization of amine 6 (78%):
mp ) 295-300 °C; 1H NMR (DMSO-d6) δ 12.05 (bs, 1H), 8.84
(d, 1H), 8.53 (d, 1H), 8.51 (d, 1H), 8.34 (d, 1H), 7.91 (t, 1H),
7.71 (t, 1H), 7.34 (m, 1H); MS (ES+) ) 197. Anal. (C12H8N2O)
C, H, N.
2-[3-(2,2-Dim et h ylp r op ion yla m in o)p yr id in -4-yl]-N,N-
d iisop r op ylben za m id e (8). Boronic acid 1 (1.3 g, 5.2 mmol)
and 2,2-dimethyl-N-(4-iodo-3-pyridinyl)propanamide (700 mg,
2.3 mmol) 7 were dissolved in DME (25 mL). Tetrakistri-
phenylphosphinepalladium (133 mg, 0.11 mmol) and 2 M
sodium carbonate solution (2.2 mL) were added. The reaction
mixture was refluxed at 83 °C for 18 h. The mixture was
concentrated in vacuo, extracted with EtOAc, washed with
brine, and dried with sodium sulfate. The crude oil was
chromatographed (CH2Cl2, 1-5% MeOH/CH2Cl2) to obtain 8
as a white solid (503 mg, 57%): 1H NMR (DMSO-d6) δ 9.08 (s,
1 H), 8.64 (s, 1 H), 8.43 (d, 1 H), 7.58-7.48 (m, 2 H), 7.40 (dd,
1 H), 7.33 (d, 1 H), 7.24 (dd, 1 H), 3.53-3.36 (m, 2 H), 1.38 (d,
3 H), 1.01 (d, 3 H), 0.97 (s, 9 H), 0.91 (d, 3 H), 0.77 (d, 3 H);
MS (ES+) ) 382.0. Anal. (C23H31N3O) C, H, N.
This material was carried on to the cyclization without
further characterization. The undesired isomer 17b was also
isolated as a yellow oil (1.01 g, 38%): 1H NMR (CDCl3) δ 8.26
(d, 1H), 7.45 (m, 4H), 7.34 (t, 1H), 3.99 (m, 1H), 3.41 (m, 1H),
3-Aza -5[H]-p h en a n th r id in -6-on e (9). Amide 8 (485 mg,
1.27 mmol) was dissolved in methanol (20 mL). Concentrated
HCl (1 mL) was added, followed by 24 h of refluxing of the
mixture. A white solid that precipitated out of solution was
filtered and dissolved in H2O. After 15 min of stirring, the free
base crashed out of solution. The solid was filtered and dried,
providing 150 mg of the desired final product 9 as a white solid
(60%): mp ) 303-309 °C; 1H NMR (DMSO-d6) δ 8.70 (s, 1
H), 8.60 (d, 1 H), 8.41 (t, 2H), 8.31 (d, 1H), 7.95 (t, 1H), 7.80
(t, 1H); MS (ES+) ) 197. Anal. (C12H8N2O‚0.3H2O) C, H, N.
5,6-Dih yd r oben zo[f][1,7]n a p h th yr id in -5-yla m in e (12).
The boronic acid 11 (1.1 g, 4.6 mmol), prepared according to
the literature method,23 2-cyano-3-bromopyridine (770 mg, 4.2
1.37 (bs, 6H), 1.21 (d, 6H); MS (ES+) ) 363. Anal. (C18H20
-
ClN3O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Nitr oa m in es
18b-g. Chloride 17a (300 mg, 0.83 mmol) was dissolved in
THF (5 mL) followed by the addition of diisopropylethylamine
(160 µL, 0.91 mmol) and 2-(4-aminoethyl)morpholine (220 µL,
1.66 mmol). The reaction mixture was heated to 65 °C
overnight, and TLC analysis indicated a low running spot on
the baseline (EtOAc). Water (5 mL) was added to the mixture

Aza-5[H]-phenanthridin-6-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3147
followed by extraction with CH2Cl2 (3 × 10 mL). The combined
organics were dried and concentrated in vacuo to yield a crude
foam that solidified upon drying in vacuo. The solid was
triturated with hexanes and filtered to yield the desired amine
18b (320 mg, 85%).
N,N-Diisop r op yl-2-[6-(2-m or p h olin -4-yleth yla m in o)-3-
n itr op yr id in -2-yl]ben za m id e (18b): yield ) 85%; 1H NMR
(DMSO-d6) δ 8.21 (m, 1H), 7.40 (m, 5H), 6.36 (d, 1H), 3.97 (m,
1H), 3.70 (m, 6H), 3.47 (m, 2H), 3.31 (m, 1H), 2.45 (m, 6H),
1.48 (bs, 3H), 1.24 (bs, 3H), 1.06 (bs, 3H), 0.87 (bs, 3H); MS
(ES+) ) 456. Anal. (C24H33N5O4) C, H, N.
2-[6-(2-Dieth yla m in oeth yla m in o)-3-n itr op yr id in -2-yl]-
N,N-d iisop r op ylben za m id e (18c): yield ) 92%; 1H NMR
(CDCl3) δ 8.15 (d, 1H), 7.39 (m, 3H), 7.30 (m, 1H), 6.26 (d,
1H), 3.96 (bs, 1H), 3.50 (bs, 1H), 3.30 (m, 1H), 2.53 (m, 3H),
1.86 (bs, 1H), 1.71 (m, 2H), 1.48 (bs, 3H), 1.35 (bs, 3H), 1.04
(t, 10H), 0.76 (bs, 3H). MS (ES+) ) 442. Anal. (C25H37N5O3) C,
H, N.
2-(6-Am in o-3-n itr op yr id in -2-yl)-N,N-d iisop r op ylben za -
m id e (18d ): yield ) 72%; 1H NMR (CDCl3) δ 8.15 (d, 1H), 7.42
(m, 2H), 7.33 (m, 2H), 6.42 (d, 1H), 5.32 (s, 2H), 3.95 (m, 1H),
3.33 (m, 1H), 1.43 (bs, 6H), 0.99 (bs, 6H). MS (ES+) ) 343.
Anal. (C18H22N4O3) C, H, N.
N,N-Diisop r op yl-2-[6-(4-isop r op ylp ip er a zin -1-yl)-3-n i-
tr op yr id in -2-yl]ben za m id e (18e): yield ) 83%; 1H NMR
(CDCl3) δ 8.19 (d, 1H), 7.33-7.43 (m, 2H), 7.25 (bs, 1H), 6.51
(d, 1H), 3.97 (bs, 1H), 3.76 (s, 4H), 3.28 (m, 1H), 2.71 (m, 1H),
2.58 (t, 4H), 1.48 (s, 3H), 1.22 (s, 3H), 1.04 (d, 6H), 1.00 (s,
3H), 0.67 (s, 3H). MS (ES+) ) 454. Anal. (C25H35N5O3) C, H,
N.
N,N-Diisop r op yl-2-(5′-n it r o-4-p yr r olid in -1-yl-3,4,5,6-
tetr ah ydr o-2H-[1,2′]bipyr idin yl-6′-yl)ben zam ide (18f): yield
) 81%; 1H NMR (CDCl3) δ 8.17 (d, 1H), 7.40 (m, 3H), 7.27 (m,
1H), 6.65 (d, 1H), 4.45 (bs, 1H), 3.97 (bs, 1H), 3.27 (m, 1H),
3.05 (t, 2H), 2.58 (s, 5H), 2.25 (m, 1H), 1.98 (d, H), 1.79 (s,
5H), 1.50 (m, 6H), 1.21 (bs, 3H), 1.00 (bs, 3H), 0.67 (bs, 3H).
MS (ES+) ) 426. Anal. (C27H37N5O3‚0.5H2O) C, H, N.
N,N-Diisop r op yl-2-[6-(4-m eth ylp ip er a zin -1-yl)-3-n itr o-
pyr idin -2-yl]ben zam ide (18g): yield ) 83%; 1H NMR (CDCl3)
δ 8.19 (d, 1H), 7.34-7.46 (m, 3H), 7.25 (m, 1H), 6.53 (d, 1H),
3.97 (s, 1H), 3.76 (bs, 4H), 3.28 (m, 1H), 2.47 (t, 4H), 2.33 (s,
3H), 1.48 (bs, 3H), 1.22 (bs, 3H), 1.00 (bs, 3H), 0.68 (bs, 3H);
MS (ES+) ) 426. Anal. (C23H31N5O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of An ilin es 19b-
g. Nitro compound 18b (300 mg, 0.66 mmol) was dissolved in
MeOH (20 mL) with Pd/C (100 mg), and the mixture was
hydrogenated at 30 psi for 2 h. TLC indicated complete
conversion of the nitro compound (10% MeOH/EtOAc). The
reaction mixture was filtered through a plug of Celite, and
the filtrate was concentrated and dried. The crude foam was
used in the cyclization step without further purification (275
mg, 99%): 1H NMR (CDCl3) δ 7.41 (m, 3H), 6.98 (d, 1H), 6.31
(d, 1H), 4.75 (bs, 2H), 3.78 (m, 1H), 3.70 (m, 4H), 3.28 (m, 3H),
2.56 (m, 2H), 2.47 (m, 4H), 1.50 (d, 3H), 1.19 (d, 3H), 1.00 (d,
3H), 0.83 (d, 3H); MS (ES+) ) 426. Anal. (C24H35N5O2) C, H,
N.
2-(5′-Am in o-4-pyr r olidin -1-yl-3,4,5,6-tetr ah ydr o-2H-[1,2′]-
bip yr id in yl-6′-yl)-N,N-d iisop r op ylben za m id e (19f): yield
) 95%; 1H NMR (CDCl3) δ 7.42 (m, 3H), 7.28 (m, 1H), 6.98 (d,
1H), 6.60 (d, 1H), 4.09 (m, 3H), 3.69 (m, 1H), 3.27 (m, 1H),
2.66 (m, 6H), 2.17 (bs, 1H), 1.98 (bd, 2H), 1.82 (s, 4H), 1.61
(m, 2H), 1.49 (d, 3H), 1.17 (d, 3H), 0.98 (d, 3H), 0.73 (d, 3H);
MS (ES+) ) 448. Anal. (C18H20N4O5‚0.25C4H8O2‚0.5H2O) C, H,
N.
2-[3-Am in o-6-(4-m eth ylpiper azin -1-yl)pyr idin -2-yl]-N,N-
d iisop r op ylben za m id e (19g): yield ) 89%; 1H NMR (CDCl3)
δ 7.42 (m, 3H), 7.26 (m, 2H), 6.97 (d, 1H), 6.57 (d, 1H), 3.68
(m, 1H), 3.37 (m, 7H), 2.51 (t, 4H), 2.33 (s, 3H), 1.48 (d, 3H),
1.15 (d, 3H), 0.96 (d, 3H), 0.68 (d, 3H); MS (ES+) ) 396. Anal.
(C23H33N5O‚C4H8O2) C, H, N.
Gen er a l P r oced u r e for t h e Cycliza t ion of An ilin es
19b-g. The crude aniline 19b (270 mg, 0.64 mmol) was
dissolved in THF (20 mL) and cooled to -78 °C. A 2.0 M
solution of LDA (1 mL) was added to the aniline, and the
reaction mixture was slowly warmed to room temperature
overnight. The mixture was quenched with water (10 mL) and
extracted several times with EtOAc (3 × 15 mL). The combined
organics were dried and concentrated and the resulting solid
was triturated with EtOAc (3 mL) and filtered, yielding the
desired amine 4b (125 mg, 58%).
2-(2-Mo r p h o lin -4-y le t h y la m in o )-5H -b e n zo [c][1,5]-
n a p h th yr id in -6-on e (4b): yield ) 58%; mp ) 250-255 °C;
1H NMR (DMSO-d6) δ 11.46 (s, 1H), 8.70 (d, 1H), 8.32 (d, 1H),
7.92 (t, 1H), 7.71 (t, 1H), 7.49 (d, 1H), 6.82 (d, 1H), 6.63 (t,
1H), 3.66 (m, 4H), 3.58 (m, 2H), 2.60 (m, 4H); MS (ES+) ) 325.
Anal. (C18H20N4O2‚0.5H2O) C, H, N.
2-(2-D ie t h y la m in o e t h y la m in o )-5H -b e n zo [c][1,5]-
n a p h th yr id in -6-on e (4c): yield ) 45%; mp ) 114-116 °C;
1H NMR (DMSO-d6) δ 11.40 (s, 1H), 8.83 (d, 1H), 8.24 (d, 1H),
7.84 (t, 1H), 7.65 (t, 1H), 7.41(d, 1H), 6.70 (d, 1H), 3.39 (m,
2H), 2.49 (m, 6H), 1.72 (m, 2H), 0.96(t. 6H); MS (ES-) ) 233.
Anal. (C19H24N4O‚0.2H2O) C, H, N.
2-Am in o-5H-ben zo[c][1,5]n aph th yr idin -6-on e (4d): yield
) 75%; mp ) 310-315 °C; 1H NMR (DMSO-d6) δ 11.42 (s,
1H), 8.50 (d, 1H), 8.26 (d, 1H), 7.85 (t, 1H), 7.66 (t, 1H), 7.44
(d, 1H), 7.70 (d, 1H), 6.02 (d, 2H); MS (ES+) ) 212. Anal.
(C12H9N3O‚0.10C4H8O2) C, H, N.
2-(4-Isop r op ylp ip er a zin -1-yl)-5H -b en zo[c][1,5]n a p h -
th yr id in -6-on e (4e): yield ) 57%; mp ) 260-264 °C; 1H NMR
(DMSO-d6) δ 11.48 (s, 1H), 8.63 (d, 1H), 8.25 (d, 1H), 7.86 (t,
1H), 7.67 (t, 1H), 7.53 (d, 1H), 7.10 (d, 1H), 3.55 (t, 4H), 2.68
(m, 1H), 2.56 (t, 4H), 1.00 (d, 6H); MS (ES+) ) 323. Anal.
(C19H24N4O) C, H, N.
2-(4-P yr r olid in -1-ylp ip er id in -1-yl)-5H -b en zo[c][1,5]-
n a p h th yr id in -6-on e h yd r och lor id e (4f‚HCl): yield ) 52%;
mp ) 170-175 °C; 1H NMR (D2O) δ 7.89 (d, 1H), 7.80 (d, 1H),
7.54 (t, 1H), 7.43 (t, 1H), 6.94 (d, 1H), 6.54 (d, 1H), 4.08 (d,
2H), 3.63 (t, 2H), 3.31 (m, 1H), 3.13 (m, 2H), 2.70 (t, 2H), 2.11-
2.2 (m, 4H), 1.94 (q, 2H), 1.63 (q, 2H). Anal. (C21H24N4O‚1H2O‚
1.4HCl) C, H, N.
2-(4-Me t h y lp ip e r a zin -1-y l)-5H -b e n zo [c][1,5]n a p h -
th yr id in -6-on e (4g): yield ) 45%; mp ) 283-285 °C; 1H NMR
(CDCl3) δ 11.50 (s, 1H), 8.65 (d, 1H), 8.27 (d, 1H), 7.88 (t, 1H),
7.69 (t, 1H), 7.56 (d, 1H), 7.14 (d, 1H), 3.56 (t, 4H), 2.46 (t,
4H), 2.24 (s, 3H). MS (ES+) ) 295. Anal. (C17H18N4O) C, H, N.
Gen er a l P r oced u r e for Sa lt F or m a tion (4g‚MsOH). The
free base 4g (1.0 g, 3.4 mmol) was suspended in hot THF (200
mL) followed by the addition of methanesulfonic acid (326 mg,
3.4 mmol). Stirring was continued overnight, and the resulting
solid was collected by vacuum filtration and dried in vacuo to
yield the mesylate salt 4g‚MsOH (1.28 g, 97%): 1H NMR (D2O)
δ 7.74 (d, 2H), 7.48 (t, 1H), 7.40 (t, 1H), 6.78 (d, 1H), 6.37 (d,
1H), 4.09 (d, 2H), 3.55 (d, 2H), 3.08 (t, 2H), 2.92-2.96 (m, 5H),
2.74 (s, 3H). Anal. (C17H18N4O‚MsOH) C, H, N.
2-[3-Am in o-6-(2-d iet h yla m in oet h yla m in o)p yr id in -2-
yl]-N,N-d iisop r op ylben za m id e (19c): yield ) 92%; 1H NMR
(CDCl3) δ 7.42 (m, 3H), 7.26 (m, 2H), 6.96 (d, 1H), 6.33 (d,
1H), 3.75 (m, 1H), 3.23 (m, 3H), 2.53 (m, 7H), 1.74 (m, 4H),
1.48 (d, 3H), 1.26 (s, 6H), 1.16 (d, 3H), 1.02 (m, 6H), 0.82 (m,
6H); MS (ES+) ) 412. Anal. (C25H39N5O1‚1.8MeOH) C, H, N.
2-(3,6-Dia m in op yr id in -2-yl)-N ,N -d iisop r op ylb e n z-
1
a m id e (19d ): yield ) 89%; H NMR (CDCl3) δ 7.43 (m, 3H),
7.28 (m, 1H), 6.98 (d, 1H), 6.45 (d, 1H), 4.07 (bs, 2H), 3.78 (m,
1H), 3.32 (m, 1H), 1.50 (d, 3H), 1.19 (d, 3H), 1.01 (d, 3H), 0.89
(d, 3H). Anal. (C18H24N4O‚0.5C4H8O2‚0.5H2O) C, H, N.
2-[3-Am in o-6-(4-isop r op ylp ip er a zin -1-yl)p yr id in -2-yl]-
N,N-d iisop r op ylben za m id e (19e): yield ) 98%; 1H NMR
(CDCl3) δ 7.42 (m, 3H), 7.28 (d, 1H), 6.96 (d, 1H), 6.56 (d, 1H),
3.68 (m, 1H), 3.35 (m, 5H), 2.63 (m, 5H), 1.46 (d, 3H), 1.15 (d,
3H), 1.08 (d, 3H), 0.95 (d, 3H), 0.68 (d, 3H); MS (ES+) ) 424.
Anal. (C25H37N5O‚0.7C4H8O2) C, H, N.
Meth od B. Syn th esis of 2-(6-Ch lor o-3-n itr op yr id in -2-
yl)ben zoic Acid E t h yl E st er (21a ). The boronic ester 20
(16.0 g, 61.0 mmol), dichloronitropyridine 16 (11.7 g, 61 mmol),
and potassium carbonate (21 g, 152 mmol) were dissolved in
toluene/EtOH (20:1, 300 mL). This mixture was purged of
oxygen and refilled several times with nitrogen. Then, tet-

3148 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Ferraris et al.
rakistriphenylphosphinepalladium (∼2 g) was added followed
by heating the mixture to 80 °C overnight. The reaction
mixture was then concentrated in vacuo and partitioned
between EtOAc (200 mL) and H2O (200 mL). The organic layer
was dried with sodium sulfate and concentrated in vacuo. The
crude residue was chromatographed using a gradient system
(5% EtOAc/hexanes to 20% EtOAc/hexanes). The final product
21a (Rf ) 0.3, 10% EtOAc/hexanes) was isolated as a low-
melting solid/foam (8.40 g, 45%): 1H NMR (CDCl3) δ 8.40 (d,
1H), 8.14 (d, 1H), 7.65 (t, 1H), 7.55 (m, 2H), 7.32 (d, 1H), 4.16
(q, 2H), 1.19 (t, 3H); MS (ES+) ) 307. Anal. (C14H11ClN2O4) C,
H, N. The undesired isomer 21b (Rf ) 0.2, 10% EtOAc/
hexanes) was also isolated (6.9 g, 35%): 1H NMR (CDCl3) δ
8.32 (d, 1H), 7.92 (d, 1H), 7.56 (m, 4H), 4.25 (dd, 2H), 1.21 (t,
3H); MS (ES+) ) 307. Anal. (C14H11ClN2O4) C, H, N.
Syn th esis of 2-[6-(4-Meth ylp ip er a zin -1-yl)-3-n itr op y-
r id in -2-yl]ben zoic Acid Eth yl Ester (22a ). The chloride 21a
(7.12 g, 23.2 mmol) was dissolved in THF (250 mL). Diisopro-
pylethylamine (3.3 g, 25.5 mmol) was added to this solution
followed by N-methylpiperazine (4.6 g, 46.4 mmol). This
mixture was heated to 60 °C overnight until complete conver-
sion of the chloride was evident by TLC (Rf of diamine ) 0.1,
EtOAc). The reaction was worked up by removal of THF and
partitioning between water (200 mL) and CH2Cl2 (200 mL).
After two more extractions with CH2Cl2 (2 × 100 mL), the
organic layer was dried with sodium sulfate and concentrated
in vacuo to yield the crude diamine 22a as a yellow oil (6.56
g, 77%): 1H NMR (CDCl3) δ 8.32 (d, 1H), 8.07 (d, 1H), 7.58 (t,
1H), 7.48 (t, 1H), 7.26 (d, 1H), 6.60 (d, 1H), 4.13 (q, 2H), 3.73
(t, 4H), 2.46 (t, 4H), 2.33 (s, 3H), 1.13 (t, 3H). MS (ES+) ) 371.
Anal. (C19H22N4O4) C, H, N.
afforded the TFA salt of 4i as an off-white solid (98%): mp )
160-165 °C; 1H NMR (DMSO-d6) δ 11.54 (s, 1H), 8.68 (d, 1H),
8.59 (d, 1H), 7.90 (t, 1H), 7.72 (t, 1H), 7.61 (d, 1H), 6.92 (d,
1H), 5.04 (s, 1H), 4.53 (s, 1H), 3.68 (m, 2H), 3.29 (m, 2H), 2.19
(d, 1H), 2.00 (d, 1H). Anal. (C17H16N4O‚1.1TFA‚0.4H2O) C, H,
N.
2-P ip er a zin -1-yl-5H -b en zo[c][1,5]n a p h t h yr id in -6-on e
1
(4j): yield ) 95%; mp ) 250-252 °C; H NMR (DMSO-d6) δ
11.56 (s, 1H), 8.93 (bs, 1H), 8.65 (d, 1H), 8.27 (d, 1H), 7.87 (t,
1H), 7.70 (t, 1H), 7.61 (d, 1H), 7.21 (d, 1H), 3.61 (t, 4H), 3.26
(bs, 4H). Anal. (C16H16N4O‚0.5H2O) C, H, N.
(S,S)-2-(5-Meth yl-2,5-d ia za bicyclo[2.2.1]h ep t-2-yl)-5H-
b en zo[c][1,5]n a p h t h yr id in -6-on e m esyla t e (4k ‚MsOH ).
This compound was made by alkylation of compound 4j as
described in the literature.35 Salt formation was performed by
suspending the free base (100 mg, 0.32 mmol) in THF (20 mL)
followed by the addition of MsOH (35 mg, 0.36 mmol). The
resulting salt precipitated out and was collected by filtration
(110 mg, 26% overall): 1H NMR (D2O) δ 8.19 (d, 1H), 8.01 (d,
1H), 7.71 (t, 1H), 7.59 (t, 1H), 7.18 (d, 1H), 6.48 (d, 1H), 4.88
(m, 1H), 4.46 (m, 1H), 3.69 (m, 2H), 3.34 (m, 2H), 2.97 (s, 3H),
2.79 (s, 3H), 2.38 (m, 2H). Anal. (C17H16N4O‚1.1MsOH‚0.4H2O)
C, H, N.
2-(4-Meth yl-4-oxypiper azin -1-yl)-5H-ben zo[c][1,5]n aph -
th yr id in -6-on e (4l). This compound was synthesized by
oxidation of 4g (200 mg, 0.68 mmol) with mCPBA (235 mg,
1.0 mmol) in dichloroethane. After the mixture was stirred
overnight, the solid that precipitated out was filitered off,
dissolved in water (5 mL), and extracted with EtOAc (3 × 10
mL). The aqueous layer was acidified with citric acid and
extracted again with EtOAc (3 × 10 mL) to remove traces of
m-chlorobenzoic acid. The remaining aqueous layer was
concentrated down, and the desired N-oxide 4l precipitated
out and was collected by filtration (158 mg, 75%): mp ) 300-
2-(3-Nit r o-6-p ip er a zin -1-ylp yr id in -2-yl)b en zoic Acid
Eth yl Ester (22b): yellow oil; yield ) 90%; 1H NMR (CDCl3)
δ 8.32 (d, 1H), 8.07 (d, 1H), 7.58 (t, 1H), 7.48 (t, 1H), 7.25 (d,
1H), 6.60 (d, 1H), 4.14 (dd, 2H), 3.72 (m, 4H), 2.97 (m, 2H),
2.57 (m, 2H), 2.50 (bs, 1H), 1.13 (t, 3H); MS (ES+) ) 357. Anal.
(C18H20N4O4) C, H, N.
1
320 °C (dec); H NMR (D2O) δ 7.63 (d, 1H), 7.53 (d, 1H), 7.32
(m, 2H), 6.60 (d, 1H), 6.16 (d, 1H), 3.79 (m, 4H), 3.60 (t, 2H),
3.55 (s, 3H), 3.08 (t, 2H); MS (ES+) ) 311. Anal. (C17H18N4O2‚
0.4H2O) C, H, N.
(S,S)-5-[6-(2-Eth oxyca r bon ylp h en yl)-5-n itr op yr id in -2-
yl]-2,5-d ia za bicyclo[2.2.1]h ep ta n e-2-ca r boxylic a cid ter t-
2-(4-Cyclopr opylm eth ylpiper azin -1-yl)-5H-ben zo[c][1,5]-
n a p h th yr id in -6-on e m esyla te (4m ‚MsOH): yield ) 82%; 1H
NMR (D2O) δ 8.55 (d, 1H), 8.17 (d, 1H), 7.76 (t, 1H), 7.58 (t,
1H), 7.46 (d, 1H), 7.05 (d, 1H), 3.49 (m, 4H), 2.40 (m, 4H), 2.12
(m, 2H), 0.77 (m, 1H), 0.39 (m, 2H), 0.00 (m, 2H). Anal.
(C20H22N4O‚1.15MsOH‚0.7H2O) C, H, N.
1
bu tyl ester (22c): yellow oil; yield ) 82%; H NMR (CDCl3)
δ 8.34 (d, 1H), 8.09 (d, 1H), 7.58 (t, 1H), 7.51 (t, 1H), 7.25 (d,
1H), 6.29 (d, 1H), 4.71 (m, 1H), 4.56 (m, 1H), 4.13 (dd, 2H),
3.54 (m, 2H), 3.40 (m, 2H), 1.91 (m, 2H), 1.43 (s, 9H), 1.14 (t,
3H); MS (ES+) ) 469. Anal. (C24H28N4O6) C, H, N.
2-[6-(4-Cyclop r op ylm eth ylp ip er a zin -1-yl)-3-n itr op yr i-
d in -2-yl]ben zoic a cid eth yl ester (22d ): yellow oil; yield
) 83%; H NMR (CDCl3) δ 8.32 (d, 1H), 8.08 (d, 1H), 7.58 (t,
1H), 7.48 (t, 1H), 7.25 (d, 1H), 6.60 (d, 1H), 4.13 (dd, 2H), 3.75
(m, 4H), 2.58 (m, 4H), 2.30 (m, 2H), 1.12 (t, 3H), 0.87 (m, 1H),
0.53 (m, 2H), 0.10 (m, 2H); MS (ES+) ) 411. Anal. (C22H26N4O4)
C, H, N.
P r oced u r e for Syn th esizin g Ch lor a cetyl Der iva tives
23 a n d 27. Amine 4d (or 4n ) was dissolved in DMA, and the
mixture was cooled to 0 °C in an ice bath. Triethylamine (1.1
equiv) and chloroacetyl chloride (0.44 mL, 5.5 mmol) were
added. The reaction mixture was stirred at room temperature
under nitrogen overnight. Solvent was evaporated under
reduced pressure, and to the resulting brown residue was
added water and 10% NaHCO3. Solid was collected by filtration
to yield chloride 23 as a white solid (1.03 g, 84%): mp ) 287-
290 °C; 1H NMR (DMSO-d6) δ 11.81 (s, 1H), 10.94 (s, 1H), 8.66
(d, 1H), 8.31 (d, 1H), 8.20 (d, 1H), 7.96 (t, 1H), 7.77 (m, 2H),
4.42 (s, 2H). Anal. (C14H10ClN3O2) C, H, N.
2-Ch lor o-N-(6-oxo-5,6-d ih yd r ob en zo[c][1,5]n a p h t h y-
r id in -3-yl)a ceta m id e (27): yield ) 87%; mp ) 280-284 °C;
1H NMR (DMSO-d6) δ 11.82 (s, 1H), 10.86 (s, 1H), 8.65 (m,
2H), 8.26 (m, 2H), 7.90 (t, 1H), 7.71 (t, 1H), 4.36 (s, 2H). Anal.
(C14H10ClN3O2) C, H, N.
Gen er a l P r oced u r e for th e Am in a tion of Ch lor id es 23
a n d 27. The chloride 23 (690 mg, 2.4 mmol) was suspended
in DMA (20 mL) followed by addition of dimethylamine (2.0
M solution in THF, 6 mL, 12 mmol). The mixture was heated
to 80 °C overnight, and the resulting mixture was stripped of
solvent and washed with water (50 mL). The resulting solid
was suspended in THF followed by addition of HCl (2.0 M in
1
Red u ction /Cycliza tion To F or m 4g. The crude diamine
22a was dissolved in MeOH (300 mL). Wet Raney nickel was
added (500 mg, catalytic amount) followed by dropwise addi-
tion of hydrazine hydrate (4.1 g, 82 mmol). The mixture was
heated to reflux and monitored by TLC until completion
(approximately 3 h). The product Rf value was 0.1 in 10%
MeOH/EtOAc. The Raney nickel was then filtered off, the
filtrate was concentrated, and the solid that resulted was
filtered off and triturated with 50 mL of CH3CN and filtered.
The resulting light-yellow solid was dried under high vacuum
for 2 h to yield 4g (4.1 g, 84%). This material was spectroscopi-
cally identical to the product isolated from method A.
(S,S)-5-(6-Oxo-5,6-d ih yd r oben zo[c][1,5]n a p h th yr id in -
2-yl)-2,5-diazabicyclo[2.2.1]h eptan e-2-car boxylic acid ter t-
1
bu tyl ester (4h ): yield ) 36%; mp ) 259-261 °C; H NMR
(DMSO-d6) δ 11.44 (s, 1H), 8.65 (d, 1H), 8.26 (d, 1H), 7.67 (t,
1H), 7.45 (d, 1H), 6.85 (t, 1H), 4.92 (dd, 2H), 3.38 (m, 2H), 3.30
(s, 1H), 3.23 (m, 1H), 1.96 (d, 2H), 1.60 (s, 9H). Anal.
(C22H24N4O3) C, H, N.
Et
2O, 1.2 mL, 2.4 mmol) and precipitation of a solid. The
resulting solid was triturated with diethyl ether and collected
via filtration to afford the desired amine 24a as a white solid
(727 mg, 82%).
2-Dim et h yla m in o-N-(6-oxo-5,6-d ih yd r ob en zo[c][1,5]-
n a p h th yr id in -2-yl)a ceta m id e h yd r och lor id e (24a ‚HCl):
(S,S)-2-(2,5-Dia za bicyclo[2.2.1]h ep t-2-yl)-5H-ben zo[c]-
[1,5]n a p h th yr id in -6-on e tr iflu or oa ceta te (4i‚TF A). This
compound was made by the deprotection of the Boc group of
compound 4e by 10% TFA/DCM (16 h). Removal of the solvent

Aza-5[H]-phenanthridin-6-ones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3149
Meth od s for Deter m in in g EC50 Va lu es. The H2O2 cyto-
mp ) 195-198 °C; 1H NMR (D2O) δ 7.87 (d, 1H), 7.76 (d, 1H),
7.56 (t, 1H), 7.47 (m, 2H), 7.06 (d, 1H), 4.15 (s, 2H), 3.00 (s,
6H). Anal. (C16H16N4O2‚1.5H2O‚1.0HCl) C, H, N.
N-(6-Oxo-5,6-d ih yd r oben zo[c][1,5]n a p h th yr id in -2-yl)-
2-p ip er id in -1-yla ceta m id e h yd r och lor id e (24b‚HCl): yield
) 87%; mp ) 175-180 °C; 1H NMR (D2O) δ 8.03 (bd, 1H),
7.90 (d, 1H), 7.66 (t, 1H), 7.59 (d, 1H), 7.54 (t, 1H), 7.22 (d,
1H), 4.12 (s, 2H), 3.63 (s, 2H), 3.13 (s, 2H), 1.86 (m, 6H). Anal.
(C19H20N4O2‚2H2O‚HCl) C, H, N.
toxicity assay was utilized for EC
50 determination as outlined
by our group in a previous publication.25 P388D1 cells (CCL-
46, ATCC, Rockville, MD), derived from murine-macrophage-
like tumor, were maintained in Dulbeco’s modified Eagle
medium (DMEM) with 10% horse serum and 2 mM L-
glutamine. The cytotoxicity assay was set up in a 96-well plate.
In each well, 190 µL cells were seeded at 2 × 106/mL density.
To determine the EC50, the concentration of a compound
required to achieve 50% reduction of cell death, a dose-
responsive experiment was conducted. The inhibitors were
added to the media with final concentrations of 0.01, 0.03, 0.1,
0.3, 1, 3, 10, and 30 µM. Each data point was an average of
quadruplicate measurements. After 15 min of incubation with
the inhibitors, 5 µL of freshly prepared H2O2 was added to the
cells to a final concentration of 2 mM. Cells were returned to
a 37 °C incubator for 4 h. At the end of the incubation, 25 µL
of supernatant were sampled from the cell media to determine
the level of lactate dehydrogenase released from dead cells.
We used an LDH assay adapted from Sigma Co. (St. Louis,
MO) and followed the experimental procedure according to the
manufacturer. The LDH activity was determined by monitor-
ing the rate of decrease of NADH absorbency at 340 nM.
Background LDH activity was subtracted. The group without
drug treatment was used to calculate total cell death due to
H2O2 treatment. The EC50 was determined from a dose-
response curve.
N-(6-Oxo-5,6-d ih yd r oben zo[c][1,5]n a p h th yr id in -2-yl)-
2-(4-p yr r olid in -1-ylp ip er id in -1-yl)a cet a m id e‚H Cl (24c‚
HCl): yield ) 92%; mp ) 196-200 °C; H NMR (D2O) δ 7.61
1
(m, 2H), 7.47 (t, 1H), 7.38 (m, 2H), 6.90 (d, 1H), 3.68 (m, 2H),
3.50 (s, 2H), 3.33 (m, 4H), 2.68 (m, 2H), 2.39 (m, 3H), 1.87-
2.12 (m, 6H). Anal. (C23H27N5O2‚2H2O‚HCl) C, H, N.
2-(4-Isop r op ylp ip e r a zin -1-yl)-N -(6-oxo-5,6-d ih yd r o-
b en zo[c][1,5]n a p h t h yr id in -2-yl)a cet a m id e h yd r och lo-
r id e (24d ‚2HCl): yield ) 90%; 1H NMR (D2O) δ 7.92 (d, 1H),
7.80 (d, 1H), 7.59 (m, 2H), 7.47 (t, 1H), 7.16 (d, 1H), 3.54 (m,
1H), 3.49 (s, 2H), 3.32 (m, 4H), 2.87 (m, 4H), 1.34 (d, 6H). Anal.
(C21H25N5O2‚1.5H2O‚2.0HCl) C, H, N.
2-(3,5-D in it r o p y r id in -2-y l)-N ,N -d iis o p r o p y lb e n z-
a m id e (26): yellow foam; yield ) 85%; 1H NMR (CDCl3) δ
9.55 (d, 1H), 9.05 (d, 1H), 7.51 (m, 2H), 7.39 (t, 2H), 4.02 (bs,
1H), 3.43 (bs, 1H), 1.33 (bs, 6H), 1.20 (bs, 6H). Anal.
(C18H20N4O5) C, H, N.
3-Am in o-5H-ben zo[c][1,5]n a p h th yr id in -6-on e (4n ). The
cyclization to form amine 4n was accomplished through the
diamine cyclization with LDA in the same manner as amine
4d (48%): mp ) 300-310 °C; 1H NMR (DMSO-d6) δ 11.46 (bs,
1H), 8.49 (d, 1H), 8.17 (d, 1H), 7.95 (s, 1H), 7.77 (t, 1H), 7.51
(t, 1H), 6.79 (s, 1H), 5.92 (d, 2H). Anal. (C12H9N3O ‚1.0H2O)
C, H, N.
Meth od s for Solu bility Testin g. The solubility data was
recorded by a Nepheloskan Ascent Instrument, type 750
manufactured by Labsystems. The instrument settings were
the following: PMT voltage ) 300; lamp voltage ) 10.0. The
first step is to prepare the vehicles in which solubility values
are desired. The following vehicles were used for evaluation:
50 mM citrate buffer (pH 4.0), 70 mM phosphate buffer saline
(PBS) (pH 7.4). Citrate buffer was prepared by dissolving 10.5
g of citric acid in 1000 mL of deionized (DI) water and
adjusting the pH using 10 N NaOH. PBS was prepared by
dissolving 1.9 g of KH2PO4, 8.1 g of Na2HPO4, and 4.1 g of
NaCl in 1000 mL of DI water and adjusting the pH using 10
N NaOH. The test compounds were dissolved in the vehicle
at 5 mg/mL. The mixtures were vortexed for 5 s, sonicated for
10 min at 37 °C, and then vortexed again for 5 s. The samples
(400 µL of each) were transferred to a 96-well plate and
immediately analyzed for turbidity.
2-Dim et h yla m in o-N-(6-oxo-5,6-d ih yd r ob en zo[c][1,5]-
n a p h th yr id in -3-yl)a ceta m id e h yd r och lor id e (28a ‚HCl):
yield ) 83%; mp ) 285-289 °C; 1H NMR (D2O) δ 7.82 (d, 1H),
7.76 (d, 1H), 7.70 (d, 1H), 7.63 (t, 1H), 7.50 (t, 1H), 7.40 (d,
1H), 4.16 (s, 2H), 3.03 (s, 6H). Anal. (C16H16N4O2‚H2O‚1.0HCl)
C, H, N.
N-(6-Oxo-5,6-d ih yd r oben zo[c][1,5]n a p h th yr id in -3-yl)-
2-p ip er id in -1-yla ceta m id e h yd r och lor id e (28b‚HCl): yield
1
) 82%; H NMR (D2O) δ 8.03 (bd, 1H), 7.90 (d, 1H), 7.66 (t,
1H), 7.59 (d, 1H), 7.54 (t, 1H), 7.22 (d, 1H), 4.12 (s, 2H), 3.63
(s, 2H), 3.13 (s, 2H), 1.86 (m, 6H). Anal. (C19H20N4O2‚H2O‚
1.0HCl) C, H, N.
Meth od s for Eva lu a tin g th e Degr a d a tion of 4g in
Micr osom a l En zym es. Compound 4g‚MsOH (100 µM) was
incubated with pooled liver microsomes (0.1 or 1.0 mg/mL)
from rat, dog, monkey, or human and with an NADPH-
generating system (1 mM NADP+, 10 mM glucose 6-phosphate,
1 U/mL glucose 6-phosphate dehydrogenase, 5 mM MgCl2) in
100 mM potassium phosphate buffer, pH 7.4. The reactions
were initiated by substrate addition. Incubations were carried
out at 37 °C. Aliquots of 300 µL were withdrawn at various
time points and mixed with 500 µL of acetonitrile to stop the
reaction. Quantitation was performed on an LC/MS/MS system
consisting of an Agilent 1100 HPLC system with a YMC basic
reversed-phase column coupled to a Micromass Ultima mass
spectrometer.
N-(6-Oxo-5,6-d ih yd r oben zo[c][1,5]n a p h th yr id in -3-yl)-
2-(4-p yr r olid in -1-ylp ip er id in -1-yl)a cet a m id e m esyla t e
(28c‚2MsOH): yield ) 89%; 1H NMR (D2O) δ 7.48 (d, 1H), 7.31
(d, 1H), 7.29 (m, 2H), 7.17 (t, 1H), 6.99 (s, 1H), 3.58 (s, 2H),
3.43-3.31 (m, 5H), 3.22 (m, 2H), 2.91 (m, 2H), 2.74 (m, 2H),
2.48 (s, 6H), 2.14 (m, 2H), 1.89-1.65 (m, 6H). Anal. (C23H27N5O2‚
2H2O‚2MsOH) C, H, N.
2-(4-Dim e t h yla m in op ip e r id in -1-yl)-N -(6-oxo-5,6-d i-
h yd r oben zo[c][1,5]n a p h th yr id in -3-yl)a ceta m id e h yd r o-
ch lor id e (28d ‚HCl): yield ) 89%; 1HNMR (D2O) δ 7.78 (d,
1H), 7.60 (m, 3H), 7.50 (t, 1H), 7.23 (s, 1H), 3.74 (m, 3H), 3.47
(s, 2H), 3.40 (m, 4H), 2.90 (m, 2H), 1.55 (d, 6H). Anal.
(C21H25N5O2‚2.25H2O‚1HCl) C, H, N.
P ARP -1 In h ibition Assa y. Purified recombinant human
PARP from Trevigan (Gaithersburg, MD) was used to deter-
mine the IC50 values of a PARP inhibitor. The PARP enzyme
assay is set up on ice in a volume of 100 µL consisting of 50
mM Tris-HCl (pH 8.0), 2 mM MgCl2, 30 µg/mL of DNase
activated herring sperm DNA (Sigma, MO), 30 µM [3H]-
nicotinamide adenine dinucleotide (67 mCi/mmol), 75 µg/mL
PARP enzyme, and various concentrations of the compounds
to be tested. The reaction is initiated by incubating the mixture
at 25 °C. After 15 min of incubation, the reaction was
terminated by adding 500 µL of ice-cold 20% (w/v) trichloro-
acetic acid. The precipitate formed is transferred onto a glass
fiber filter (Packard Unifilter-GF/B) and washed three times
with ethanol. After the filter is dried, the radioactivity is
determined by scintillation counting.
Meth od s for Eva lu a tin g P h a r m a cok in etic Sa m p les of
4g, 4l, 4j, 4m , a n d 24c. The selected PARP-1 inhibitors were
dosed as an intravenous bolus into male Wistar rats at 10 mg/
kg over 5 min (5 mL total volume). One animal was sacrificed
at each time point, and samples from the desired tissues or
plasma were collected and assayed as described below.
Sa m p le P r ep a r a tion . The calibration standards covered
the range 1-1000 ng/m. The quality control (QC) standard
ranges were 3, 30, 300, and 800 ng/mL. A 1:10 dilution of the
800 ng/mL QC sample was run. Sample from tissue, calibra-
tion standards, and QC samples were subjected to protein
precipitation by acetonitrile followed by filtration in an Anasys,
Captiva 96-well filter plate. All samples were analyzed on a
LC/MS/MS (MicroMass Ultima) with an electrospray source
in positive ion mode.

3150 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Ferraris et al.
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An a lytica l Meth od for 4g. Mobile phase: isocratic, ac-
etonitrile (ACN), and 0.1% formic acid in water. Column: YMC
Basic, 3 µm, 4.0 mm × 50.0 mm. Injection volume: 10 µL.
An a lytica l Meth od for 4j. Mobile phase: gradient of ACN
and 10 mM NH4AC (ammonium acetate), 15-75% ACN linear
gradient from 0 to 2.0 min was run, followed by isocratic 75%
ACN until 2.4 min. From 2.4 to 4.0 min, the column was
reequilibrated at 15% ACN. Column: Phenomenex Aqua, 3
µm C18 125A, 30 mm × 4.60 mm. Injection volume: 10 µL.
An a lytica l Meth od for 4m . Mobile phase: gradient of
ACN and 10 mM NH4AC (ammonium acetate), 15-75% ACN
linear gradient from 0 to 2.0 min was run, followed by isocratic
75% ACN until 2.4 min. From 2.4 to 4.0 min, the column was
reequilibrated at 15% ACN. Column: Phenomenex Aqua, 3
µm C18 125A, 30 mm × 4.60 mm. Injection volume: 20 µL.
An a lytica l Meth od for 4l. Mobile phase: gradient of ACN
and 10 mM NH4AC (ammonium acetate), 15-75% ACN linear
gradient from 0 to 2.0 min was run, followed by isocratic 75%
ACN until 2.4 min. From 2.4 to 4.0 min, the column was
reequilibrated at 15% ACN. Column: YMC Basic, 3 µm, 4.0
mm × 50.0 mm. Injection volume: 10 µL.
An a lytica l Meth od for 24c. Mobile phase: isocratic,
acetonitrile (ACN), and 0.1% formic acid in water. Column:
YMC Cyno, 3 µm 120A, 30 mm × 50.0 mm. Injection volume:
25 µL.
Meth od for Testin g P ARP In h ibitor s in F oca l Cer e-
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modified by Belayev.31 Male Sprague-Dawley rats (280-320
g, Charles River, Wilmington, MA) were anesthetized with
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the internal carotid artery into the origin of the middle cerebral
artery (MCA). The suture is removed after 2 h of MCA
occlusion.
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Permanent focal cerebral ischemic stroke was modeled in
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Dawley, Indianapolis, IN) were anesthetized with isofluorane.
The right and left common carotid arteries and the right distal
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ized, and the carotids were transiently occluded for 90 min.36
All drugs were administered 30 min after the start of MCA
occlusion. For both models, the brains were collected into cold
PBS at 24 h after ischemia. The brains were sliced and
processed for TTC histochemistry, and the apparent infarcts
were measured using computer-assisted planimetry. In the
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