Novel tricyclic poly(ADP-ribose) polymerase-1 inhibitors with potent anticancer chemopotentiating activity: Design, synthesis, and X-ray cocrystal structure

Article abstract of DOI:10.1021/jm020259n

A series of novel compounds have been designed that are potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1), and the activity and physical properties have been characterized. The new structural classes, 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol

Full text of DOI:10.1021/jm020259n

J . Med. Chem. 2002, 45, 4961-4974
4961
Expedited Articles
Novel Tr icyclic P oly(ADP -r ibose) P olym er a se-1 In h ibitor s w ith P oten t
An tica n cer Ch em op oten tia tin g Activity: Design , Syn th esis, a n d X-r a y Cocr ysta l
Str u ctu r e
Stacie S. Canan Koch,*,† Lars H. Thoresen,‡,§ J ayashree G. Tikhe,† Karen A. Maegley,† Robert J . Almassy,†,|
J ianke Li,†, Xiao-Hong Yu,† Scott E. Zook,†,# Robert A. Kumpf,† Cathy Zhang,† Theodore J . Boritzki,†
Rena N. Mansour,†,∇ Kanyin E. Zhang,†,∇ Anne Ekker,† Chris R. Calabrese,‡ Nicola J . Curtin,‡ Suzanne Kyle,‡
Huw D. Thomas,‡ Lan-Zhen Wang,‡ A. Hilary Calvert,‡ Bernard T. Golding,‡ Roger J . Griffin,‡ David R. Newell,‡
Stephen E. Webber,†,[ and Zdenek Hostomsky†
Pfizer Global Research and Development, La J olla/ Agouron Pharmaceuticals, Inc., 10770 Science Center Drive,
San Diego, California 92121, and University of Newcastle, Newcastle upon Tyne, NE2 4HH, U.K.
Received J une 19, 2002
A series of novel compounds have been designed that are potent inhibitors of poly(ADP-ribose)
polymerase-1 (PARP-1), and the activity and physical properties have been characterized. The
new structural classes, 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-ones and 3,4-dihydro-
pyrrolo[4,3,2-de]isoquinolin-5-(1H)-ones, have conformationally locked benzamide cores that
specifically interact with the PARP-1 protein. The compounds have been evaluated with in
vitro cellular assays that measure the ability of the PARP-1 inhibitors to enhance the effect of
cytotoxic agents against cancer cell lines.
In tr od u ction
rapid synthesis and degradation of ADP polymers can
result in abrupt and profound cellular NAD+ depletion.
Poly(ADP-ribose) polymerase-1 (PARP-1: EC 2.4.2.30)
is the most abundant and best characterized member
of a family of PARP enzymes with important cellular
functions.1-6 PARP-1, a nuclear enzyme implicated in
the maintenance of genomic integrity, consists of three
functional domains: an N-terminal DNA-binding do-
main, a C-terminal catalytic domain, and a central
automodification domain. PARP-1 binds to and is acti-
vated by DNA strand breaks and catalyzes the synthesis
of homopolymers of ADP-ribose from NAD+ onto nuclear
acceptor proteins. PARP-1 itself is the main protein
acceptor (automodification), but the enzyme has also
been shown to modify histones, topoisomerases, DNA
polymerases, and ligases.7,8 The formation of these
negatively charged polymers in the vicinity of the DNA
nick is thought to cause the electrostatic repulsion of
PARP-1 (and histones) from the DNA and to facilitate
the recruitment of the base excision repair (BER)
complex.9 The ADP-ribose polymers formed by PARP
are cleaved by the cellular hydrolase poly(ADP-ribose)
glycohydrolase (PARG). PARP-1 activation and the
Cytotoxic drugs or radiation can induce activation of
PARP-1, and it has been demonstrated that inhibitors
of PARP-1 can potentiate the DNA damaging and
cytotoxic effects of chemotherapy and irradiation.10
Because of the essential function of PARP-1 for cellular
repair and survival, we sought to evaluate the potential
of novel, potent PARP-1 inhibitors for the treatment of
cancer in combination with selected cytotoxic agents.
Inhibitors have been designed to mimic the substrate-
protein interactions of NAD+ with PARP-1. The inhibi-
tors are theorized to bind to PARP-1 and to prevent
polymer formation and hence the electrostatic repulsion
of PARP from DNA, thus inhibiting the repair of the
DNA strand breaks. This leads to eventual cell death.
Early PARP inhibitors were analogues of 3-amino-
benzamide (Figure 1).11 It was realized that the benz-
amide functionality was critical for specific binding to
PARP-1, and analogue synthesis indicated that the
s-trans conformation of the benzamide was the most
favorable binding conformation for PARP inhibition.12
Recent cocrystal structures have confirmed this critical
binding mode.13 PARP-1 inhibitors have since been
designed to lock the benzamide group in this specific
s-trans conformation.14 Some examples of PARP-1 in-
hibitors that incorporate the locked conformation at the
amide functionality, with either bicyclic systems (NU1025
and PD128763) or intramolecular hydrogen bonding
arrangements (NU1085), are shown in Figure 1. Al-
though some of these PARP-1 inhibitors displayed
potent in vitro activity, they lacked specificity, had poor
physical properties, had unfavorable pharmacokinetic
* To whom correspondence should be addressed. Phone: (858) 622-
7504. Fax: (858) 526-4151. E-mail: stacie.canan@pfizer.com.
† Pfizer Global Research and Development, La J olla/Agouron Phar-
maceuticals, Inc.
‡ University of Newcastle.
§ Current address: Department of Chemistry, Texas A&M Univer-
sity, College Station, TX.
| Current address: Quorex Pharmaceuticals, San Diego, CA 92121.
Current address: 8937 Gainsborough Avenue, San Diego, CA
92129.
#
Current address: Neurocrine Pharmaceuticals, San Diego, CA
92121.
∇ Current address: Novartis Genomics Institute, La J olla, CA 92121.
[ Current address: Anadys Pharmaceuticals, San Diego, CA 92121.
10.1021/jm020259n CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/15/2002

4962 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Canan Koch et al.
F igu r e 1. Examples of reported PARP-1 inhibitors.
Sch em e 1. Key Tricyclic Intermediates
F igu r e 2. Model of newly designed tricyclic PARP-1 inhibi-
tors.
Sch em e 2. Synthesis of 2-Halo Tricyclic Indoles
profiles, or had in vivo side effects that prevented them
from being developed further as clinical agents.
Design
It was our objective to design more potent PARP-1
inhibitors with favorable physical properties and in vivo
profiles suitable for preclinical development. We pro-
posed novel tricyclic indole analogues that would struc-
turally restrict the benzamide rotation and allow the
optimum interactions with the protein active site (Fig-
ure 2). The new PARP-1 inhibitors were designed with
a benzamide functionality that is locked in the s-trans
conformation via a ring connection (i.e., lactam) to an
indole core. Modeling studies indicated that these
tricyclic systems would allow maximum interaction
(three critical hydrogen bonds) of the lactam carbonyl/
N-H with the glycine-863 and serine-904 residues at
the protein active site. Two tricyclic structures are
reported here: the 3,4,5,6-tetrahydro-1H-azepino[5,4,3-
cd]indol-6-ones ([5,6,6]-tricylic indole lactams) and the
3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5-(1H)-ones([5,6,7]-
tricylic indole lactams). The tricyclic design allows for
maximum incorporation of diversity by functionalization
at the C-2 position of the indole ring. Both the six- and
seven-membered lactams were investigated, and we
predicted from the modeling studies that the [5,6,7]-
system would exhibit the most favorable physical prop-
erties (solubility, melting point) and still participate in
productive protein interactions with PARP-1 (vide in-
fra).
Sch em e 3. Palladium-Catalyzed Coupling Reactions To
Give 2-Aryl Tricyclic Indolesa
a
Ar defined in Tables 2 and 6.
tricyclic indole intermediate, 3, was also iodinated at
the 2-position with bis[(trifluoroacetoxy)iodo]benzene
and iodine to give the 2-iodo-[5,6,6]-tricyclic indole, 6
(Scheme 2). Each tricyclic bromide underwent Suziki-
type couplings (Scheme 3) with a variety of arylboronic
acids to give the 2-aryl-[5,6,7]- or [5,6,6]-tricyclic indole
lactams (7-19), which were evaluated for PARP-1
activity. Formyl substituted 2-aryl tricyclic indole lac-
tams (20-23) were further functionalized through
reductive amination with sodium cyanoborohydride and
a variety of primary and secondary amines (24-28,
Scheme 4).
To investigate the critical nature of the lactam
functionality in the tricyclic inhibitors, several ana-
logues were prepared to disrupt the hydrogen bonding
ability of this group. Scheme 5 illustrates the synthesis
of four analogues: methylation (NaH, MeI) of the indole
nitrogen to give 29 and the lactam nitrogen to yield the
dimethyl analogue 30; conversion to the thiolactam 31
Ch em istr y
The synthesis of the tricyclic indole lactams utilizes
the Leimgruber-Batcho indole cyclization to generate
the key 4-carboxyindole intermediate (1, Scheme 1) and
essentially follows procedures that have been pub-
lished.15,16 The [5,6,6]-tricyclic core, 2, was prepared as
described in the literature by Demerson et al.17 (Scheme
1). The [5,6,7]-tricyclic core, 3, was prepared from the
4-carboxyindole by procedures described by Flaugh,
Clark, Bowman, and Somei.18,19
The tricyclic intermediates (2 and 3) were brominated
at the 2-position with pyridinium tribromide to yield the
tricyclic bromides 4 and 5 (Scheme 2). The [5,6,7]-

Poly(ADP-ribose) Polymerase-1 Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4963
Sch em e 4. Reductive Amination Procedure To Yield
apparent in X-ray cocrystal structures of representative
analogues from the [5,6,7]-tricyclic series (compounds
7 and 25) with the PARP-1 protein (chicken PARP-1
catalytic domain, cPARP-1 CD; see Figures 3 and 4).
The critical hydrogen bonds between the lactam func-
tionality and the Gly-863 and Ser-904 residues in this
tricyclic series of compounds are apparent in both
cocrystal structures, and ideal hydrogen bond distances
have been established between the ligand and the
protein (2.7-3.0 Å). The nonplanar conformation of the
seven-membered lactam most likely contributes to this
ideal interaction between the tricyclic ligands and the
protein, allowing the lactam carbonyl and -NH to adopt
a position closer to the protein residues involved in the
hydrogen bonding. Additionally, the cocrystal structure
indicates that nearby tyrosine residues (Tyr-907, Tyr-
889, and Tyr-896) in the protein likely participate in
π-π-type interactions (pseudo-edge-to-face and pseudo-
face-to-face) with the indole core and the C-2 aryl
substituent. Both structures reveal an ordered water
molecule that participates in hydrogen bonding (2.8-
3.1 Å) between the indole NH and the important
catalytic Glu-988 residue.20 Gln-763 appears to be
mobile, and the movement of this residue nicely accom-
modates the p-benzylamine substituent in compound 25.
The cocrystal X-ray structure of the amine compound,
25 (Figure 4), displays other unique changes in the
protein conformation. The hydrogen bond between
residues Tyr-889 and Asp-766 (see Figure 3) seen in the
structure of the C-2 unsubstituted phenyl analogue, 7,
with the PARP-1 protein appears to be broken in the
25 cocrystal structure where the p-dimethylamino-
Benzylaminesa
a
R1 and R2 defined in Tables 3 and 6.
with Lawesson’s reagent; and reduction of the lactam
carbonyl with LAH to give the azatricycle 32.
The 2-iodotricycle (6) was converted into the 2-carboxy
or 2-carbonitrile derivatives by palladium-mediated
reactions (Scheme 6). Finally, 2-carbamide analogues
were generated from the 2-carboxy intermediate (33) by
treatment with methylamine in methanol or coupling
(HATU) of the acid (34) with other primary amines.
Cocr ysta l Str u ctu r es
Cocrystal structures of the new tricyclic PARP-1
inhibitors indicate that the lactam functionality can
adopt an ideal conformation for multiple interactions
with the PARP-1 protein. These specific interactions are
Sch em e 5. Analogues Designed To Evaluate Lactam Interactions with PARP-1 Protein
Sch em e 6. Synthesis of 2-Carboxy and 2-Carbonitrile Tricyclic Indoles

4964 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Canan Koch et al.
F igu r e 3. Cocrystal structure (2.3 Å resolution) of compound 7 and cPARP-1 CD protein.
F igu r e 4. Cocrystal structure (2.3 Å resolution) of compound 25 and cPARP-1 CD protein.
methyl substituent is occupying this space. In fact, the
entire loop between Gly-888 and Gly-892 moves away
from the ligand to accommodate the alkylamino group.
Finally, as seen in Figure 4, an extra water molecule is
apparent in the binding site and is situated in the face
of the C-2 aryl ring of compound 25.
Tyne, U.K.) or topotecan (TP; SmithKline Beecham
Pharmaceuticals, Philadelphia, PA) for 5 days with or
without coexposure to 0.4 µM PARP-1 inhibitor.20 The
0.4 µM inhibitor concentration displays no intrinsic
growth inhibitory activity. At the end of treatments, the
relative cell number was determined by the SRB as-
say.21 Data were calculated as a percentage of drug-free
(1% DMSO) control or PARP inhibitor alone control. Cell
growth IC50 values (concentration of drug required to
inhibit cell growth by 50%) were calculated from the
generated growth inhibition curves using point-to-point
analysis (GraphPad Software, Inc., San Diego, CA). To
quantify the potentiation of the cytotoxicity of topotecan
(TP) or temozolomide (TM) by test compounds, a di-
mensionless parameter PF50, the potentiation factor at
50% growth inhibition, was defined as the ratio of IC50
of the control (cytotoxic drug TM or TP alone) to IC50 of
the sample (cytotoxic drug plus PARP-1 inhibitor com-
Meth od s
Gr ow th In h ibition Assa ys. A549 cells (human lung
carcinoma, American Type Culture Collection (ATCC),
Rockville, MD, or National Cancer Institute, NIH,
Bethesda, MD), SW620 cells (human colon carcinoma,
ATCC), or LoVo cells (human colorectal cancer, ATCC)
were seeded in 96-well plates for 16-24 h before
experimental manipulation. To determine the extent of
chemosensitization, cells were continuously exposed to
increasing concentrations of temozolomide (TM; a gift
from the Cancer Research Campaign, Newcastle upon

Poly(ADP-ribose) Polymerase-1 Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4965
Ta ble 1. PARP-1 Inhibition Observed for [5,6,7]-Tricyclic Indolesa
a
Ki values are averages of at least two independent experiments. Experimental variation in Ki values was less than 20% for all
compounds.
Ta ble 2. PARP-1 Activity of Substituted 2-Aryl Tricyclic
Indoles
bination). A PF50 value of 1.0 indicates no effect. Data
are normalized to DMSO as a control or to 0.4 µM
PARP-1 inhibitor alone as a control.
To determine the intrinsic growth inhibitory proper-
ties of the compounds in comparison to the minimum
concentration required to maximally potentiate TM and
TP, cells (LoVo or SW620) grown in 96-well plates were
exposed to increasing concentrations of PARP inhibitor
in the absence or presence of a concentration of cytotoxic
drug that caused <50% inhibition of cell growth, i.e.,
400 µM TM, 10 nM TP (LoVo cells), or 2 nM TP (SW620
cells) for 5 days and cell growth was determined by the
SRB assay. As appropriate, data were normalized to
DMSO control or to TM or TP alone as controls.
NAD+ Dep letion Assa y. A549 cells were treated
with MNNG for 20 min in the presence or absence of
PARP inhibitors. After the treatment, cells were washed
once with 1X Hank’s balanced saline solution and
extracted with 20% trichloroacetic acid (TCA). The TCA
insoluble fraction was removed by centrifugation. The
TCA supernatant was extracted with water-saturated
ethyl ether to remove TCA. Following removal of TCA,
NAD was determined by an enzymatic cycling assay as
described,22 and the ADP-ribose polymers were quanti-
fied as described.23
compd
R
Ki (µM)
7
8
9
H
0.006
0.008
0.023
0.032
0.006
0.029
<0.005
0.005
0.263
0.007
0.006
0.230
2-Cl
2-OH
2-SMe
4-OMe
4-tBu
10
11
12
13
14
15
16
17
18
4-F
4-CF3
4-CO2H
3-NH2
3-CF3
3,5-diCF3
nM) retains potent PARP-1 activity; however, the 1,5-
N,N-dimethyl analogue (30, Ki g 10 µM) is a weak
PARP-1 inhibitor. This indicates that the lactam N-H
participates in a beneficial interaction with the PARP-1
protein. Additional evidence for the role of the lactam
functionality is supported by the relatively weak activity
of the thiolactam (31, Ki ) 57 nM) and the lack of
activity for the azatricycle (32, Ki g 10 µM). Compound
7 has been cocrystallized with the PARP-1 protein
(cPARP-1 CD; see Figure 3). The X-ray structure does
show specific interactions of the lactam carbonyl with
Ser-904 and Gly-863 and of the lactam -NH with Gly-
863. Additionally, the 2-phenyl group may participate
in π-π-type interactions with Tyr-907 and Tyr-896 (see
Figure 3).
Representative examples of substituted 2-aryl tricyclic
indoles and the corresponding PARP-1 enzymatic activ-
ity are shown in Table 2. Aryl groups with ortho
substituents have reduced PARP-1 activity relative to
the other regioisomers, and the activity decreases as the
size of the group increases (compounds 8-10), with the
2-thiomethyl (10) being a relatively weak PARP-1
Resu lts a n d Discu ssion
The compounds have been evaluated with an enzy-
matic activity assay (see Experimental Section, Ki,
Tables 1-6) and with in vitro cellular assays that
measure the ability of the PARP-1 inhibitors to enhance
the effect of cytotoxic agents against cancer cell lines
(PF50 values; see Tables 7 and 8). Ki values are averages
of at least two independent experiments. Experimental
variation in Ki values was less than 20% for all
compounds. The 2-phenyl tricyclic indole (7, Ki ) 6 nM)
is approximately 6 times more potent in terms of Ki than
the unsubstituted indole (3, Ki ) 38 nM), suggesting
that the C-2 aryl group is interacting favorably at the
PARP-1 binding site. The 1-N-methylindole (29, Ki ) 7

4966 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Canan Koch et al.
Ta ble 3. PARP-1 Activity of Amino-Substituted 2-Aryl
Tricyclic Indoles
Ta ble 5. PARP-1 Activity of 2-Carboxy and Carboxamide
Tricyclic Indoles
compd
R
Ki (µM)
compd
R
Ki (µM)
36
37
38
33
34
NH-Me
NH-iPr
NH-Ph
OMe
0.014
0.032
0.010
0.010
0.370
24
25
26
3-CH2NMe2
4-CH2NMe2
4-CH2-(1-pyrrolidinyl)
0.008
0.005
0.006
OH
Ta ble 4. PARP-1 Activity of Various 2-Substituted Tricyclic
Indoles
Ta ble 6. PARP-1 Activity of [5,6,6]-Tricyclic Indoles
R
compd
C-2 substituent
Ki (µM)
compd
R
Ki (µM)
35
39
40
41
42
43
44
CN
0.011
0.007
0.005
0.023
0.005
0.008
0.009
2
19
27
28
H
Ph
0.047
<0.005
<0.005
<0.005
3-pyridinyl
1-naphthyl
2-biphenyl
2-thiophene
2-pyrrolyl
5-indolyl
(3-CH2NMe2)Ph
(4-CH2NMe2)Ph
Ta ble 7. Cytotoxicity Potentiation of Topotecan (PF50(TP)) in
A549 Cells by PARP-1 Inhibitors
compd
Ki (µM)
PF50(TP) a
inhibitor (Ki ) 32 nM). The larger ortho substituent may
force the 2-phenyl ring to rotate into an unfavorable
conformation that disrupts the binding affinity to PARP-
1. Aryl groups substituted at the para position retain
PARP-1 activity, similar to the parent (compounds 11,
13, and 14; Ki e 5-6 nM). Notable is the slight loss in
activity with the para tert-butyl substituent (12, Ki )
29 nM) that indicates a steric restriction at this end of
the binding site. The 4-carboxy analogue (15, Ki ) 263
nM) displays comparatively poor PARP-1 activity, pos-
sibly due to a repulsive interaction of the negatively
charged carboxylate with the protein. Aryl groups with
single meta substituents (compounds 16 and 17; Ki )
6-7 nM) retain good activity; however, the 3,5-bis-CF3
analogue (18) displays poor PARP-1 activity (Ki ) 230
nM). Analysis of this structure with computational
models indicates that there are some unfavorable steric
interactions with residues of the protein near the active
site. Overall, the PARP-1 binding site appears to be
fairly tolerant of a variety of functional groups at each
phenyl regioisomer in these new tricyclic inhibitors.
Although the physical properties of the new tricyclic
inhibitors were much improved relative to the previ-
ously reported bicyclic benzimidazoles, we still sought
to enhance the properties by incorporation of an ioniz-
able group on the C-2 aryl substituent. To further
enhance the solubility, we explored various amine
substituents. Both meta and para aminomethyl substi-
tuted 2-aryl tricyclic indole inhibitors retain very favor-
able PARP-1 activity (Table 3, compounds 24-26; Ki )
5-8 nM). As an example of the improved solubility
profile, compound 25 exhibits a water solubility >800
times higher (3.2 mM, pH 7) than the water solubility
of the unsubstituted phenyl analogue 7 (0.026 mM, pH
7).
3
7
0.038
0.006
0.007
1.1
1.8
1.7
1.2
2.3
2.1
2.4
2.0
2.2
29
30
13
24
25
26
28
>10
<0.005
0.008
0.005
0.006
<0.005
a
PF50 is the ratio of IC50 of TP alone to IC50 of TP + PARP-1
inhibitor (0.4 µM).
protein could accommodate additional functionality at
the C-2 position of the indole core. In fact, other C-2
substitution was permissible, including heteroaryls,
alkyls, alkynes, carboxamides, and carboxy analogues
(Tables 4 and 5). All of the C-2 aryl and heteroaryl
substituents shown in Table 4 retain potent-to-moderate
PARP-1 activity (39-44; Ki ) 5-23 nM). Similarly, the
C-2 cyano-substituted tricycle, 35, retains PARP-1
activity in the enzymatic assay (Ki ) 11 nM).
The 2-carboxamide substituted tricyclic indoles ex-
hibit variable PARP-1 activity. Small amides (methyl,
36; Ki ) 14 nM) and the aromatic amide (38, Ki ) 10
nM) retain good enzymatic activity; however, a larger
alkyl-substituted amide (37; Ki ) 32 nM) shows some-
what reduced PARP-1 activity. The 2-carboxy-substi-
tuted tricyclic indoles exhibit even more variable activ-
ity. The methyl ester retains potent activity (33; Ki )
10 nM); however, the carboxylic acid (34; Ki ) 370 nM)
is a fairly weak PARP-1 inhibitor.
Representative analogues in the [5,6,6]-tricyclic indole
class of PARP-1 inhibitors are shown in Table 6. This
class of tricyclic PARP-1 inhibitors displays the same
activity trends as those discussed above for the [5,6,7]-
tricyclic indole series. The unsubstituted [5,6,6]-tricycle
(2; Ki ) 47 nM) exhibits moderate PARP-1 activity, and
addition of C-2 aromatic substituents greatly enhances
Examination of the X-ray cocrystal structures of the
tricyclic inhibitors with PARP-1 indicated that the

Poly(ADP-ribose) Polymerase-1 Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4967
Ta ble 8. Cellular PARP-1 Activity Influences NAD+ and ADP-Polymer Levelsa
b
monitored
cellular activity
DMSO
(control)
MNNG alone
MNNG + 7
(0.4 µM)
MNNG + 25
(0.4 µM)
MNNG + 30
(0.4 µM)
(25 µM)
NAD+ levels, %
% ADP polymer formation
100
5
21
100
81
13
87
6
24
105
a
MNNG ) 1-methyl-3-nitro-1-nitrosoguanidine, DNA damaging agent; A549 cell line. PARP-1 Ki values (µM): for 7, Ki ) 0.006; for
b
25, Ki ) 0.005; for 30, Ki ) 10. Negative control (i.e., very weak PARP-1 inhibitor).
Ta ble 9. Cytotoxicity Potentiation of Topotecan (TP) and
Temozolomide (TM) by Compound 26 in Various Cell Lines
IC50 cytotoxic
cell
line
cytotoxic agent alone
IC50 of TP or TM
agenta
(TP or TM)
and 26
PF50
b
A549
TP
TP
TP
TM
24 ( 1.0 nM
3.2 ( 0.4 nM
11.6 ( 2.0 nM
595 ( 75 µM
12 ( 0.5 nM
1.8 ( 0.2 nM
6.5 ( 0.4 nM
76 ( 2.3 µM
2.0 ( 0.1
1.8 ( 0.06
1.8 ( 0.2
7.8 ( 1
SW620
LoVo
LoVo
a
b
TP ) topotecan; TM ) temozolomide. PF50 ) (IC50 of cyto-
toxic agent alone)/(IC50 of cotherapy (cytotoxic agent plus 0.4 µM
26)). Data are mean ( SEM of three independent experiments.
PARP-1 potency. The C-2 phenyl [5,6,6]-tricycle (19; Ki
e 5 nM) and the two amino-substituted analogues (27
and 28; Ki e 5 nM) are fairly potent PARP-1 inhibitors.
The ability of these new tricyclic PARP-1 inhibitors
to enhance the effects of chemotherapy agents (topo-
tecan (TP) or temozolomide (TM)) is demonstrated here
with in vitro cellular growth inhibition assays and the
calculation of a potentiation factor (PF50) in various cell
lines. A standard concentration of 400 nM PARP-1
inhibitor, which is not intrinsically growth inhibitory,
was used. A value of PF50 greater than 1.0 indicates that
the PARP-1 inhibitor is capable of enhancing the effects
(i.e., further inhibiting cancer cell growth) of the cyto-
toxic agents (topotecan or temozolomide, Tables 7 and
8). A PF50 value of 1.0 indicates no effect. For this new
F igu r e 5. Potentiation of temozolomide (TM) cytotoxicty
(growth inhibition) by compound 26 in LoVo cells. Data,
normalized to 1% DMSO or 0.4 µM 26 alone as control, are
from a single representative (three total) experiment.
the depletion of NAD+ or the production of ADP polymer
after treatment with MNNG, similar to the response
observed with no PARP-1 inhibitor treatment.
Additional in vitro potentiation experiments were
conducted with compound 26, and the results in various
cell lines are summarized in Table 9. This PARP-1
inhibitor, 26, is capable of enhancing the effects (reduced
cell survival) of both topotecan and temozolide in three
separate cells lines (A549, LoVo, and SW690) by factors
ranging from 1.8-fold to 7.8-fold.10g Figure 5 illustrates
the profound 7.8-fold enhancement of TM-induced growth
inhibition achieved by cotreatment with compound 26
(0.4 µM) in LoVo cells.
Further in vitro experiments illustrate the growth
inhibitory (IC50) effect of PARP-1 inhibitor 26 alone and
with a sub-IC50 concentration of temozolomide (TM) or
topotecan (TP). LoVo cells were exposed to increasing
concentrations of PARP inhibitor 26 in the absence or
presence of 400 µM temozolomide or 10 nM topotecan
(Figure 6). High concentrations (>1 µM) of 26 exhibit
some intrinsic growth inhibition, but at concentrations
that were not intrinsically growth inhibitory, compound
26 significantly increased the temozolomide- and topo-
tecan-induced growth inhibition. Indeed, near-maximal
potentiation was achieved at 0.1 µM 26, a concentration
at least 10× lower than the concentration of 26 alone,
causing measurable growth inhibition.
class of PARP-1 inhibitors, the potentiation factor (PF50
)
correlates with the Ki data (Table 7). Generally, the
PARP-1 inhibitors with a Ki less than 10 nM exhibited
good potentiation of TP (PF50 > 1.7). Compounds 7 and
29 potentiate or enhance the effect of topotecan in A549
cells by 1.8 and 1.7 times, respectively. This experiment
indicates that the IC50 value of topotecan/PARP-1
inhibitor combination therapy is enhanced relative to
topotecan alone in this cell line. (Compound 7 alone is
relatively nontoxic with an IC50 of 12 µM.) The 4-F
analogue (13) has a favorable potentiation factor (PF50
) 2.3), and this may reflect some increase permeability
in the cellular assay. As a class, the amino-substituted
tricyclic PARP-1 inhibitors (compounds 24-26, 28,
Table 7) also displayed favorable potentiation factors
with topotecan in A549 cells (PF50 ) 2.0-2.4). The
weaker PARP-1 inhibitors (3 and 30) display little or
no potentiation of the cytotoxic agent (PF50 ) 1.1-1.2).
A functional assay of PARP-1 activity in A549 cells
(which monitors PARP-1-mediated NAD+ depletion in
cells exposed to DNA alkylating agents) clearly dem-
onstrates that the new compounds inhibit PARP-1
activity in intact cells (Table 8). Cells that are damaged
by MNNG and simultaneously treated with the PARP-1
inhibitors, 7 or 25, are protected from NAD+ depletion
following the damage. The absence of ADP-polymer
formation in these cells after DNA damage and cotreat-
ment with the PARP-1 inhibitor confirms the functional
inhibition of PARP-1 by compounds 7 and 25. Con-
versely, the weak PARP-1 inhibitor 30 does not prevent
Con clu sion
A potent new class of tricyclic lactam indole PARP-1
inhibitors have been designed, and an efficient synthesis
has been realized. The binding conformation has been
evaluated with X-ray cocrystal structures of the ligands
and the PARP-1 protein. This new class of PARP-1
inhibitors, represented by compounds 7, 25 and 26,
displays potent in vitro PARP-1 enzymatic inhibition
and potent enhancement of cellular growth inhibition.

4968 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Canan Koch et al.
Concentration was calculated using the extinction coefficient
determined in 5% DMSO/water.
P u r ifica tion a n d Cr ysta lliza tion of Ch ick en P ARP -1-
CD. The expression, purification, and crystallization of the
chicken PARP-1 catalytic domain (cPARP-1-CD) were per-
formed essentially as described with minor modifications.28
Following purification by affinity chromatography using a
3-aminobenzamide affi-gel 10 column, the protein was dialyzed
overnight in 50 mM Tris, pH 7.5, 1 mM â-mercaptoethanol,
and 1 mM Pefa-bloc (buffer D). The enzyme was further
purified by passage over a DEAE-sephacryl column equili-
brated in buffer D. The protein does not bind to the column
under these conditions, but the remaining major impurity does.
The column is washed with two column volumes of buffer D.
The flow through and washes were combined, glycerol was
added to a final concentration of 20%, and the protein was
quick-frozen and stored at -70 °C until needed.
Frozen aliquots of cPARP-1-CD were thawed and concen-
trated 10-fold (to approximately 10 mg/mL) using an Amicon
centriprep-10 concentrator. The concentrated protein was
diluted with 14 mM â-mercaptoethanol back to the original
volume and concentrated again 10-fold. This process was
performed twice to remove the glycerol and Pefa-bloc. cPARP-
1-CD (6 mg/mL) was combined with 200 µM PARP-1 inhibitor,
centrifuged, and added in a 1:1 ratio with the reservoir solution
containing 100 mM Tris, pH 8.5, 8% 2-propanol, and 16-30%
PEG 600. Crystallization trials were performed at ambient
temperature using the hanging drop vapor diffusion method.
Gen er a l Exp er im en ta l P r oced u r e. All solvents were
reagent grade or better and used as supplied, unless otherwise
indicated. As required, starting materials are azeotropically
dried prior to use and reactions were conducted with standard
precautions taken to exclude moisture and oxygen. Flash
chromatography was accomplished using silica gel (Kieselgel
60, 230-400 mesh). Radial chromatography was performed
on a Harrison Research chromatotron with 1, 2, or 4 mm silica
gel coated rotors. Thin-layer chromatography utilized Analtech
250 µm plates with F-256. NMR spectra were recorded in
DMSO-d6, unless otherwise noted, on a GE 300 or a Bruker
Advance 300DPX spectrometer. IR spectra were recorded on
a Perkin-Elmer 1600 FTIR spectrometer. MS were performed
by the Scripps Research Institute MS Facility, at UC Riverside
MS Facility, or on an HP 1100 LC/MSD system. Elemental
analyses were determined by Atlantic Microlab Inc., Norcross,
GA, or Galbraith Laboratories, Nashville, TN. Melting points
were taken on a Laboratory Devices melting point apparatus
and are reported uncorrected. The tricyclic indole intermedi-
ates 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-one (3) and
3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5-(1H)-one (2) were pre-
pared from methyl indole-4-carboxylate by modification of
existing procedures.15-17
Temozolomide (TM) (a gift from the Cancer Research
Campaign, Newcastle upon Tyne, U.K.) and topotecan (TP)
(SmithKline Beecham Pharmaceuticals, Philadelphia, PA)
were dissolved in DMSO at 10 and 2.2 mM, respectively, and
stored as aliquots at -20 °C. PARP-1 inhibitors were prepared
in DMSO at 100 mM and stored at -20 °C. Drugs (alone or in
combination) were added to cell cultures so that final DMSO
concentrations were consistently 1% (v/v).
2-Br om o-3,4-dih ydr opyr r olo[4,3,2-de]isoqu in olin -5-(1H)-
on e (Com p ou n d 4). Pyridinium tribromide (90%, 0.267 g,
0.75 mmol) was added to a suspension of the [5,6,6]-tricycle 2
(0.086 g, 0.5 mmol) in 40 mL of CH2Cl2 at 0 °C. The reaction
mixture was stirred at 0 °C for 30 min. The solvent was
removed in vacuo, and ice/water was added to the residue. The
resulting suspension was stirred vigorously at 0 °C for 30 min
and then filtered to give 0.068 g (54%) of a brown solid that
was used without further purification. IR (KBr) 3172, 1655,
1606, 1441, 1367, 1292, 755 cm-1; 1H NMR (300 MHz, DMSO-
d6) δ 4.61 (s, 2H), 7.17 (app t, J ) 6 Hz, 1H), 7.32 (d, J ) 6 Hz,
1H), 7.39 (d, J ) 6 Hz, 1H), 7.71 (s, 1H), 11.92 (s, 1H); LRMS
(M + H) 251/253.
F igu r e 6. Growth inhibition by compound 26 and TM or TP
in LoVo cells. Data are normalized to 1% DMSO, 400 µM TM,
or 10 nM TP alone as control, as appropriate.
Compound 26 with TP displays 1.8- to 2.0-fold enhance-
ment of cellular growth inhibition in several cell lines
and a 7.8-fold enhancement of TM-mediated cellular
growth inhibition in LoVo cells. The tricyclic analogues
also exhibit potent PARP-1 inhibition in a functional
assay and are able to inhibit the formation of ADP
polymer and to prevent depletion of NAD+ after cellular
insult with a chemotherapy agent. The use of this new
class of compounds for potentiation of radiation and
chemotherapy is being investigated in in vivo tumor
models.24
Exp er im en ta l Section
P ARP -1 P r otein Assa y Meth od . Test compounds were
incubated in Tris buffer, pH 8.0, with 10 mM MgCl2, 1 mM
TCEP as a reducing agent, 20 nM hPARP-1 protein, 10 µg/
mL DNase I activated calf thymus DNA (Sigma), 500 µM
NAD+, and [32P]-NAD+ (0.1-0.3 µCi/rxn) at 25 °C for 4 min.25,26
The reaction was stopped by adding an equal volume of 40%
TCA and incubating the mixture on ice for 30 min. The
samples were transferred to a Bio-Dot microfiltration ap-
paratus (BioRad), filtered through Whatman GF/C glass fiber
filter paper, washed 3 times with 5% TCA and 1% PPi in
water, and dried. [32P]-ADP-ribose incorporation into the acid-
insoluble material was quantified using a phosphor imager
(Molecular Dynamics). Inhibition constants (Ki) were calcu-
lated by nonlinear regression analysis.27 Quantitation of Ki
values below 5 nM was not possible because of the sensitivity
limitations of the assay. Ki values are averages of at least two
independent experiments. Experimental variation in Ki values
was less than 20% for all compounds.
Deter m in a tion of Ma xim u m Lin ea r Solu bility. The
solubility of PARP inhibitors was determined in 5% DMSO/
water spectrophotometrically. Concentrated (20×) stocks were
prepared in 100% DMSO and diluted with distilled water to a
final DMSO concentration of 5%. The solutions were allowed
to equilibrate for 30 min at room temperature prior to removal
of any precipitated inhibitor by centrifugation. The UV-vis
spectrum for each sample was recorded, and the absorbance
maximum of a selected peak was plotted as a function of
theoretical inhibitor concentration. The linear portion of this
curve is used to calculate the extinction coefficient of the PARP
inhibitor using Beer’s law. The maximum linear solubility is
determined graphically as the maximal inhibitor concentration
in the linear range of the described plot. Solubility in 100%
water was determined by stirring a saturated solution of
compound in deionized water for 4 h at room temperature (∼22
°C). Prior to addition of compound, the water was adjusted to
pH 7.5 by the addition of Tris buffer, pH 7.5, to a final
concentration of 1 mM. The solution was then filtered to
remove solid material and analyzed on the spectrophotometer.
2-Br om o-3,4,5,6-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ol-
6-on e (Com p ou n d 5). 3,4,5,6-Tetrahydro-1H-azepino[5,4,3-

Poly(ADP-ribose) Polymerase-1 Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4969
cd]indol-6-one (3, 264 mg, 1.42 mmol) in CH2Cl2 (30 mL) and
THF (30 mL) was treated with pyridinium tribromide (534 mg,
1.67 mmol) at 0 °C. The orange solution was stirred for 10
min and then was allowed to warm to ambient temperature
and stirred for an additional hour. Water (30 mL) was added,
and the organic solvents were removed in vacuo. The aqueous
solution was adjusted to pH 8-9 with 1 M NaOH and
extracted with CH2Cl2 (3 × 30 mL). The organic solution was
washed with water and brine, dried (Na2SO4), filtered, and
concentrated. The crude product was recrystallized (CH2Cl2/
MeOH) to yield the tricyclic bromide 5 as a yellow solid: 305
added, and the mixture was stirred at 80 °C under an argon
atmosphere for 2 h. 2-Bromo-1,3,4,5,9a,9b-hexahydroazepino-
[5,4,3-cd]indol-6-one (5, 239 mg, 0.90 mmol), a second portion
of 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium (24
mg, 0.03 mmol), and sodium carbonate (2.5 mL of a 2.0 M
aqueous solution, 5.00 mmol) were then added, and the
reaction mixture was stirred under an argon atmosphere at
80 °C for another 17 h. The reaction mixture was then poured
into 25 mL of water and then was extracted with 25% isopropyl
alcohol/CHCl3 (3 × 20 mL). The combined organic extracts
were dried (MgSO4) and concentrated in vacuo, leaving a
brown oil. The crude product was passed through a short silica
plug with 25% MeOH/CHCl3 and then purified by radial
chromatography eluting with 20% MeOH/CHCl3. Crystalliza-
tion from MeOH/CHCl3/hexanes yielded 2-(2-hydroxyphenyl)-
1,3,4,5-tetrahydroazepino[5,4,3-cd]indol-6-one, 40 mg (15%),
as a white solid: mp 305 °C (dec); 1H NMR (300 MHz, DMSO-
d6) δ 2.86 (m, 2H), 3.46 (m, 2H), 6.92 (t, J ) 7.5 Hz, 1H), 7.00
(d, J ) 7.8 Hz, 1H), 7.16 (t, J ) 7.8 Hz, 1H), 7.24 (m, 1H),
7.34 (dd, J ) 7.5, 1.2 Hz, 1H), 7.55 (d, J ) 7.8 Hz, 1H), 7.66
(d, J ) 7.5 Hz, 1H), 8.00 (br t, 1H), 9.84 (br s, 1H), 11.20 (br
s, 1H); MS (FAB, MH+) 279. Anal. (C17H14N2O2‚0.44CHCl3) C,
H, N.
1
mg (81%); mp 204-206 °C (dec); H NMR (300 MHz, DMSO-
d6) δ 2.85 (m, 2H), 3.45 (m, 2H), 7.25 (t, J ) 7.8 Hz, 1H), 7.52
(d, J ) 7.8 Hz, 1H), 7.72 (d, J ) 7.8 Hz, 1H), 8.14 (br t, 1H),
1
12.05 (br s, 1H). H NMR (300 MHz, CDCl3/MeOH-d4) δ 2.81
(m, 2H), 3.45 (m, 2H), 7.08 (t, J ) 7.7 Hz, 1H), 7.34 (d, J ) 7.7
Hz, 1H), 7.70 (d, J ) 7.7 Hz, 1H). HRMS (FAB, MH+) calcd
for C11H10BrN2O: 264.9976. Found: 264.9984.
2-Iod o-1,3,4,5-t e t r a h yd r oa ze p in o[5,4,3-cd ]in d ol-6-
on e (Com p ou n d 6). 1,3,4,5-Tetrahydroazepino[5,4,3-cd]indol-
6-one (3, 620 mg, 3.35 mmol) was suspended in 80 mL of THF/
CH2Cl2 (1:1) and then cooled in an ice bath. Bis[(trifluoro-
acetoxy)iodo]benzene (1.73 g, 4.02 mmol) and iodine (850 mg,
3.35 mmol) were added, and the reaction mixture was stirred
at 0 °C for 25 min. The ice bath was removed, and the reaction
mixture was allowed to stir for another 30 min as it warmed
to room temperature. The reaction was quenched by addition
of aqueous sodium bisulfite. The layers were separated, and
the organic layer was dried over MgSO4, filtered, and concen-
trated in vacuo leaving a yellow solid. The crude solid was
purified by flash chromatography (5% MeOH/CHCl3) to yield
2-iodo-1,3,4,5-tetrahydroazepino[5,4,3-cd]indol-6-one, 6, 308
2-(2-Meth ylsu lfa n ylp h en yl)-1,3,4,5-tetr a h yd r oa zep in o-
[5,4,3-cd ]in d ol-6-on e (Com p ou n d 10). In a manner similar
to that described for compound 7, the tricyclic bromide (5, 530
mg, 2.00 mmol) and 2-thioanisole boronic acid (370 mg, 2.20
mmol) were coupled to yield 2-(2-methylsulfanylphenyl)-1,3,4,5-
tetrahydroazepino[5,4,3-cd]indol-6-one, 264 mg (43%), as an
1
off-white solid: mp 271-272 °C; H NMR (300 MHz, DMSO-
d6) δ 2.39 (s, 3H), 2.73 (m, 2H), 3.37 (m, 2H), 7.23 (m, 2H),
7.37 (m, 2H), 7.49 (m, 2H), 7.70 (d, J ) 7.2 Hz, 1H), 8.05 (br
t, 1H), 11.41 (br s, 1H); MS (FAB, MH+) 309. Anal. (C18H16N2-
OS) C, H, N.
1
mg (30%), as a pale-yellow solid. H NMR (300 MHz, DMSO-
d6) δ 2.79 (m, 2H), 3.40 (m, 2H), 7.14 (app t, J ) 7.8 Hz, 1H),
7.46 (dd, J ) 7.8, 0.6 Hz, 1H), 7.64 (dd, J ) 7.5, 0.9 Hz, 1H),
8.06 (br t, 1H), 11.80 (br s, 1H); MS (FAB, MH+) 313.
2-(4-Met h oxyp h en yl)-3,4,5,6-t et r a h yd r o-1H -a zep in o-
[5,4,3-cd ]in d ol-6-on e (Com p ou n d 11). The tricyclic bromide
(5, 48 mg, 0.18 mmol) in toluene (5 mL) and EtOH (2.5 mL)
was treated with solid Na2CO3 (48 mg, 0.45 mmol), LiCl (23
mg, 0.54 mmol), p-methoxyphenylboronic acid (41 mg, 0.27
mmol), and water (0.25 mL). The solution was degassed, and
tetrakis(triphenylphosphine)palladium(0) (10 mg, 5 mol %)
was added. The solution was heated at reflux for 13 h and
then was cooled to ambient temperature and diluted with
water (10 mL). The aqueous layer was adjusted to pH 7-8
with saturated aqueous K2CO3 and extracted with EtOAc (10
mL × 3). The organic solution was washed with water and
brine, dried (Na2SO4), filtered, and concentrated. The crude
product was recrystallized (MeOH/THF) to yield the 2-(p-
methoxyphenyl)tricycle as a white solid, 47.4 mg (89%): mp
2-P h en yl-3,4,5,6-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ol-
6-on e (Com p ou n d 7). The tricyclic bromide (5, 200 mg, 0.75
mmol) in toluene (20 mL) and EtOH (10 mL) was treated with
solid Na2CO3 (199 mg, 1.88 mmol), phenylboronic acid (138
mg, 1.13 mmol), and water (0.50 mL). The solution was
degassed, and tetrakis(triphenylphosphine)palladium(0) (43
mg, 5 mol %) was added. The solution was heated at reflux
for 5 h and then was cooled to ambient temperature and
diluted with water (20 mL). The aqueous layer was adjusted
to pH 7-8 with saturated aqueous K2CO3 and extracted with
EtOAc (20 mL × 3). The organic solution was washed with
water and brine, dried (Na2SO4), filtered, and concentrated.
The crude product was recrystallized (CH2Cl2/MeOH/hexanes)
to yield the 2-phenyltricycle, 7, as a pale-yellow solid, 183 mg
(93%): mp 249-255 °C (dec); 1H NMR (300 MHz, CDCl3/
MeOH-d4) δ 3.14 (m, 2H), 3.53 (m, 2H), 7.23 (t, J ) 7.7 Hz,
1H), 7.33 (m, 1H), 7.44 (m, 2H), 7.55 (m, 3H), 7.83 (d, J ) 7.7
Hz, 1H). HRMS (FAB, MH+) calcd for C17H15N2O: 263.1184.
Found: 263.1189. Anal. (C17H14N2O‚0.8H2O) C, H, N.
1
143-148 °C (dec); H NMR (300 MHz, DMSO-d6) δ 3.08 (m,
2H), 3.38 (m, 2H), 3.87 (s, 3H), 7.14 (d of ABq, J ) 8.6 Hz,
2H), 7.22 (t, J ) 7.5 Hz, 1H), 7.57 (d, J ) 7.5 Hz, 1H), 7.64 (d
of ABq, J ) 8.6 Hz, 2H), 7.70 (d, J ) 7.5 Hz, 1H), 8.11 (br t,
1H), 11.52 (br s, 1H). HRMS (FAB, MH+) calcd for
C
18H17N2O2: 293.1290. Found: 293.1301. Anal. (C18H16N2O2)
C, H, N.
2-(4-ter t-Bu tylp h en yl)-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-
2-(2-Ch lor op h en yl)-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]-
in d ol-6-on e (Com p ou n d 8). In a manner similar to that
described for compound 7, the tricyclic bromide (5, 210 mg,
0.79 mmol) and 2-chlorophenylboronic acid (136 mg, 0.87
mmol) were coupled to yield 2-(2-chlorophenyl)-1,3,4,5-tetra-
hydroazepino[5,4,3-cd]indol-6-one, 78 mg (33%), as a shiny
cd ]in d ol-6-on e (Com p ou n d 12). As described for compound
7, the tricyclic bromide (5, 300 mg, 1.13 mmol) and 4-tert-
butylphenylboronic acid (302 mg, 1.70 mmol) were coupled to
yield 2-(4-tert-butylphenyl)-1,3,4,5-tetrahydroazepino[5,4,3-cd]-
indol-6-one, 150 mg (42%), as a white solid: mp 243-244 °C;
1H NMR (300 MHz, DMSO-d6) δ 1.33 (s, 9H), 3.05 (m, 2H),
3.38 (m, 2H), 7.20 (app t, J ) 7.8 Hz, 1H), 7.57 (m, 5H), 7.67
(dd, J ) 7.2, 0.6 Hz, 1H), 8.07 (br t, 1H), 11.51 (br s, 1H).
HRMS (FAB, MH+) calcd for C21H23N2O: 319.1810. Found:
319.1813. Anal. (C21H22N2O‚0.3H2O) C, H, N.
2-(4-Flu or oph en yl)-3,4,5,6-tetr ah ydr o-1H-azepin o[5,4,3-
cd ]in d ol-6-on e (Com p ou n d 13). As described for compound
7, the tricyclic bromide (5, 100 mg, 0.54 mmol) and 4-fluoro-
benzeneboronic acid (79 mg, 0.57 mmol) were coupled to yield
2-(4-fluorophenyl)-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]-
indol-6-one, 107 mg (99%), as a pale-yellow solid: 1H NMR
(300 MHz, DMSO-d6) δ 3.04 (m, 2H), 3.38 (m, 2H), 7.22 (app
1
white solid: mp 275 °C (dec); H NMR (300 MHz, DMSO-d6)
δ 2.76 (m, 2H), 3.38 (m, 2H), 7.23 (apparent t, J ) 7.8 Hz,
1H), 7.56 (m, 5H), 7.71 (dd, J ) 7.5, 0.9 Hz, 1H), 8.07 (br t,
1H), 11.53 (br s, 1H); MS (FAB, MH+) 297. Anal. (C17H13N2-
OCl‚0.15H2O) C, H, N.
2-(2-Hyd r oxyp h en yl)-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-
cd ]in d ol-6-on e (Com p ou n d 9). In a manner similar to that
described by Giroux, Han, and Prasit,29 2-iodophenol (220 mg,
1.00 mmol), pinacol diborane (279 mg, 1.10 mmol), 1,1′-bis-
(diphenylphosphino)ferrocenedichloropalladium (24 mg, 0.03
mmol), and potassium acetate (294 mg, 3.00 mmol) were
combined in a Schlenk tube. The vessel was evacuated and
then refilled with argon thrice. Degassed DMF (6 mL) was

4970 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Canan Koch et al.
t, J ) 7.7 Hz, 1H), 7.39 (m, 2H), 7.56 (dd, J ) 8.0, 0.9 Hz, 1H),
7.64 (m, 3H), 8.05 (br t, 1H), 11.57 (br s, 1H). HRMS (FAB,
MH+) calcd for C17H14FN2O: 281.1090. Found: 281.1093. Anal.
(C17H13FN2O‚0.6H2O) C, H, N.
the desired product as a yellow solid: mp 215-218 °C (dec);
1H NMR (300 MHz, DMSO-d6) δ 5.04 (s, 2H), 7.17 (t, 1H, J )
7.5 Hz), 7.34 (d, 1H, J ) 6.6 Hz), 7.35 (d, 1H, J ) 7.4 Hz),
7.50 (m, 4H), 7.66 (d, 1H, J ) 7.7 Hz), 7.84 (s, 1H), 11.64 (s,
1H); HRMS (M + H) 249.1023.
3-F or m ylp h en yl-3,4-d ih yd r op yr r olo[4,3,2-d e]isoqu in o-
lin -5-(1H)-on e (Com p ou n d 20). A procedure similar to that
described for compound 21 (vide infra) was used to prepare
the target aldehyde in 23% yield: 1H NMR (300 MHz, DMSO-
d6) 5.10 (s, 2H), 7.20-7.23 (m, 1H), 7.36 (d, 1H, J ) 6.0 Hz),
7.49 (d, 1H, J ) 6.0 Hz), 7.75-7.77 (m 1H), 7.86-7.89 (m, 2H),
7.97 (d, 1H, J ) 6.0 Hz), 8.18 (s, 1H), 10.09 (s, 1H), 11.85 (s,
1H). The aldehyde was used without further purification.
2-(4-Tr iflu or om eth ylph en yl)-1,3,4,5-tetr ah ydr oazepin o-
[5,4,3-cd ]in d ol-6-on e (Com p ou n d 14). As described for
compound 7, the tricyclic bromide (5, 300 mg, 1.13 mmol) and
4-trifluoromethylphenylboronic acid (322 mg, 1.70 mmol) were
coupled to yield 2-(4-trifluoromethylphenyl)-1,3,4,5-tetra-
hydroazepino[5,4,3-cd]indol-6-one, 261 mg (70%), as an off-
1
white solid: mp 208-209 °C; H NMR (300 MHz, DMSO-d6)
δ 3.09 (m, 2H), 3.40 (m, 2H), 7.27 (app t, J ) 7.8 Hz, 1H), 7.60
(dd, J ) 8.1, 0.9 Hz, 1H), 7.71 (dd, J ) 7.5, 0.6 Hz, 1H), 7.88
(m, 4H), 8.13 (br t, 1H), 11.77 (br s, 1H); MS (FAB, MH+) 331.
Anal. (C18H13F3N2O‚1.0H2O) C, H, N.
4-F or m ylp h en yl-3,4-d ih yd r op yr r olo[4,3,2-d e]isoqu in o-
lin -5-(1H )-on e (Com p ou n d 21). To a suspension of the
bromide, 4, in 30 mL of toluene and 15 mL of EtOH was added
4-formylbenzeneboronic acid (0.457 g, 3.05 mmol), Na2CO3
(0.538 g, 5.08 mmol) dissolved in a minimum amount of water,
LiCl (0.258 g, 6.09 mmol), and tetrakistriphenylphosphine-
palladium(0) (0.117 g, 0.102 mmol). The reaction mixture was
refluxed for 48 h. The solvent was removed in vacuo and the
residue was taken up in EtOAc and washed with saturated
aqueous NaHCO3, H2O, and brine. The organic layer was dried
over MgSO4 and concentrated to give a yellow solid that was
purified by flash column chromatography eluting with a
gradient of 60-80% of EtOAc in CHCl3 to give 0.370 g of a
mixture of the acetal and the aldehyde. The acetal was
converted to the desired product using 5 mL of MeOH in 3
mL of H2O and a catalytic amount of concentrated H2SO4 to
give the aldehyde in 66% yield: IR (KBr) 1694, 1653, 1601,
4-(6-Oxo-3,4,5,6,9a ,9b-h exa h yd r o-1H-a zep in o[5,4,3-cd ]-
in d ol-2-yl)ben zoic Acid (Com p ou n d 15). In a manner
similar to that described for compound 7, the tricyclic bromide
(5, 530 mg, 2.00 mmol) and 4-carboxyphenylboronic acid (365
mg, 2.20 mmol) were coupled to yield 4-(6-oxo-3,4,5,6,9a,9b-
hexahydro-1H-azepino[5,4,3-cd]indol-2-yl)benzoic acid, 340 mg
(56%), as a pale-yellow solid: mp 345.5-346.5 °C (dec); 1H
NMR (300 MHz, DMSO-d6) δ 3.10 (m, 2H), 3.40 (m, 2H), 7.25
(t, J ) 7.8 Hz, 1H), 7.59 (dd, J ) 8.1, 0.9 Hz, 1H), 7.70 (dd, J
) 7.5, 0.6 Hz, 1H), 7.78 (m, 2H), 8.10 (m, 3H), 11.73 (br s,
1H), 13.00 (br s, 1H); MS (electrospray, MH+) 307. Anal.
(C18H14N2O3‚0.9H2O) C, H, N.
2-(3-Am in oph en yl)-3,4,5,6-tetr ah ydr o-1H-azepin o[5,4,3-
cd ]in d ol-6-on e (Com p ou n d 16). As described for compound
7, the tricyclic bromide (5, 428 mg, 1.61 mmol) and 3-amino-
benzeneboronic acid monohydrate (300 mg, 1.94 mmol) were
coupled to yield 2-(3-aminophenyl)-3,4,5,6-tetrahydro-1H-aze-
pino[5,4,3-cd]indol-6-one, 110 mg (25%), as an off-white solid:
1H NMR (300 MHz, DMSO-d6) δ 3.03 (m, 2H), 3.39 (m, 2H),
5.24 (s, 2H), 6.59 (br d, 1H), 6.78 (d, J ) 7.7 Hz, 1H), 6.84 (m,
2H), 7.18 (m, 2H), 7.52 (d, J ) 7.9 Hz, 1H), 7.66 (d, J ) 7.4
Hz, 1H), 8.04 (br t, 1H), 11.41 (br s, 1H). HRMS (FAB, MH+)
1
1261, 821, 746 cm-1; H NMR (300 MHz, DMSO-d6) 5.09 (s,
2H), 7.26 (t, J ) 6 Hz, 1H), 7.36 (d, J ) 6 Hz, 1H), 7.50 (d, J
) 6 Hz, 1H), 7.85 (d, J ) 9 Hz, 2H), 7.91 (s, 1H), 8.02 (d, J )
9 Hz, 2H), 10.01 (s, 1H), 11.86 (s, 1H); LRMS (M + H) 277.
The aldehyde was used without further purification.
2-(3-For m ylph en yl)-3,4,5,6-tetr ah ydr o-1H-azepin o[5,4,3-
cd ]in d ol-6-on e (Com p ou n d 22). As described for compound
7, the tricyclic bromide (5, 381 mg, 1.44 mmol) and 3-formyl-
benzeneboronic acid (345 mg, 2.16 mmol) were coupled to yield
2-(3-formylphenyl)-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]-
indol-6-one, 346 mg (83%), as a tan solid: 1H NMR (300 MHz,
DMSO-d6) δ 2.86 (m, 2H), 3.16 (m, 2H), 7.01 (t, J ) 7.8 Hz,
1H), 7.34 (d, J ) 7.3 Hz, 1H), 7.50 (m, 2H), 7.73 (m, 2H), 7.85
(br t, 1H), 7.94 (s, 1H), 9.88 (s, 1H), 11.50 (br s, 1H). The
aldehyde was used without further purification.
calcd for
C17H16N3O: 278.1293. Found: 278.1297. Anal.
(C17H15N3O‚1.1H2O) C, H, N.
2-(3-Tr iflu or om eth ylph en yl)-1,3,4,5-tetr ah ydr oazepin o-
[5,4,3-cd ]in d ol-6-on e (Com p ou n d 17). As described for
compound 7, the tricyclic bromide (5, 300 mg, 1.13 mmol) and
3-trifluoromethylphenylboronic acid (322 mg, 1.70 mmol) were
coupled to yield 2-(3-trifluoromethylphenyl)-1,3,4,5-tetra-
hydroazepino[5,4,3-cd]indol-6-one, 300 mg (80%), as a pale-
yellow solid: mp 212.5-213.5 °C; 1H NMR (300 MHz, DMSO-
d6) δ 3.08 (m, 2H), 3.40 (m, 2H), 7.27 (app t, J ) 7.8 Hz, 1H),
7.60 (d, J ) 7.8 Hz, 1H), 7.71 (d, J ) 7.5 Hz, 1H), 7.77 (m,
2H), 7.96 (m, 2H), 8.13 (br t, 1H), 11.78 (br s, 1H); MS (FAB,
MH+) 331. Anal. (C18H13F3N2O‚0.5H2O) C, H, N.
2-(3,5-Bistr iflu or om eth ylph en yl)-1,3,4,5-tetr ah ydr oaze-
p in o[5,4,3-cd ]in d ol-6-on e (Com p ou n d 18). As described for
compound 7, the tricyclic bromide (5, 300 mg, 1.13 mmol) and
3,5-bistrifluoromethylphenylboronic acid (202 mg, 1.24 mmol)
were coupled to yield 2-(3,5-bistrifluoromethylphenyl)-1,3,4,5-
tetrahydroazepino[5,4,3-cd]indol-6-one, 70 mg (16%), as a pale-
yellow solid: mp 230 °C (dec); 1H NMR (300 MHz, DMSO-d6)
δ 3.11 (m, 2H), 3.42 (m, 2H), 7.31 (app t, J ) 7.8 Hz, 1H), 7.64
(d, J ) 8.1 Hz, 1H), 7.73 (d, J ) 7.5 Hz, 1H), 8.13 (br s, 1H),
8.16 (br t, 1H), 8.28 (br s, 2H), 11.95 (br s, 1H); MS (FAB,
MH+) 399. Anal. (C19H12F6N2O‚0.2 hexanes) C, H, N.
2-P h en yl-3,4-d ih yd r op yr r olo[4,3,2-d e]isoq u in olin -5-
(1H)-on e (Com p ou n d 19). To a suspension of 4 (0.1065 g,
0.424 mmol) in 20 mL of toluene and 10 mL of EtOH was
added phenylboronic acid (0.08 g, 0.636 mmol), Na2CO3 (0.113
g, 1.06 mmol) dissolved in a minimum amount of water, LiCl
(0.054 g, 1.27 mmol), and tetrakis(triphenylphosphine)-
palladium(0) (24.5 mg, 21.0 µmol). The reaction mixture was
refluxed for 16 h. The solvent was removed in vacuo, and the
residue was taken up in EtOAc and washed with saturated
aqueous NaHCO3, H2O, and brine. The organic layer was dried
over Na2SO4 and concentrated to give a yellow solid, which
was purified by flash column chromatography eluting with a
gradient of 20% of EtOAc in hexanes to give 0.098 g (93%) of
2-(4-For m ylph en yl)-3,4,5,6-tetr ah ydr o-1H-azepin o[5,4,3-
cd ]in d ol-6-on e (Com p ou n d 23). As described for compound
7, the tricyclic bromide (5, 168 mg, 0.63 mmol) and 4-formyl-
benzeneboronic acid (142 mg, 0.95 mmol) were coupled to yield
2-(4-formylphenyl)-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]-
indol-6-one, 141 mg (77%), as a yellow solid: mp 238-240 °C
1
(dec); H NMR (300 MHz, DMSO-d6) δ 3.12 (m, 2H), 3.42 (m,
2H), 7.28 (t, J ) 7.6 Hz, 1H), 7.59 (d, J ) 7.6 Hz, 1H), 7.62 (d,
J ) 7.6 Hz, 1H), 7.88 (d of ABq, J ) 7.7 Hz, 2H), 8.05 (d of
ABq, J ) 7.7 Hz, 2H), 8.11 (br t, 1H), 10.07 (s 1H), 11.75 (br
s, 1H). HRMS (FAB, MH+) calcd for C18H15N2O2: 291.1134.
Found: 291.1132. The aldehyde was used without further
purification.
2-(3-(N,N-Dim et h yla m in o)m et h ylp h en yl)-3,4,5,6-t et -
r ah ydr o-1H-azepin o[5,4,3-cd]in dol-6-on e (Com pou n d 24).
The meta aldehyde 22 (346 mg, 1.19 mmol) in MeOH (40 mL)
was treated with dimethylamine (2 M solution in MeOH, 7.16
mmol). The solution was cooled with an ice/water bath and
treated dropwise with a solution of sodium cyanoborohydride
(82 mg, 1.31 mmol) and zinc chloride (89 mg, 0.66 mmol) in
MeOH (10 mL). The resulting solution was adjusted to pH 6-7
with 2 M methanolic HCl. After the mixture was stirred for
30 min, the reaction was quenched with concentrated HCl (0.2
mL) and the methanol was removed by evaporation. The
residue was diluted with water (30 mL). The solution was
adjusted to pH 10-11 with KOH(s) and extracted with CH2Cl2
(30 mL × 3). The organic solution was washed with water and
brine, dried (Na2SO4), filtered, and concentrated. The crude
product was crystallized (CH2Cl2/MeOH/hexanes) to give 2-(3-

Poly(ADP-ribose) Polymerase-1 Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4971
(N,N-dimethylamino)methylphenyl)-3,4,5,6-tetrahydro-1H-
azepino[5,4,3-cd]indol-6-one, 332 mg (87%), as shiny yellow
crystals: mp 222-225 °C; 1H NMR (300 MHz, DMSO-d6) δ
2.20 (s, 6H), 3.06 (m, 2H), 3.40 (m, 2H), 3.50 (s, 2H), 7.21 (t, J
) 7.7 Hz, 1H), 7.41 (br d, J ) 7.4 Hz, 1H), 7.50 (m, 4H), 7.69
(d, J ) 7.1 Hz, 1H), 8.05 (br t, 1H), 11.56 (br s, 1H). HRMS
(FAB, MH+) calcd for C20H22N3O: 320.1763. Found: 320.1753.
Anal. (C20H21N3O‚0.25H2O) C, H, N.
6 Hz, 1H), 7.42 (d, J ) 6 Hz, 1H), 7.48 (d, J ) 9 Hz, 2H), 7.63
(d, J ) 9 Hz, 2H), 7.81 (s, 1H), 11.62 (s, 1H); LRMS (M + H)
306; Anal. (C19H19N3O‚0.75H2O) C, H, N.
1-N-Met h yl-2-p h en yl-3,4,5,6-t et r a h yd r o-1H -a zep in o-
[5,4,3-cd ]in d ol-6-on e (Com p ou n d 29). A solution of the
tricyclic 2-phenylindole (compound 7, 51.3 mg, 0.20 mmol) in
THF (1 mL) and DMPU (0.1 mL) was cooled with an ice/water
bath and treated dropwise with a suspension of NaH (0.45
mmol) in THF (0.5 mL). The yellow mixture was allowed to
stir at 0 °C for 10 min and was treated dropwise with a 1 M
solution of MeI in THF (0.22 mL, 0.22 mmol). The mixture
was allowed to warm to ambient temperature and stirred for
30 min. The reaction was quenched at 0 °C with saturated
aqueous NH4Cl and extracted with EtOAc (5 mL × 3). The
organic solution was washed with water and brine, dried
(Na2SO4), filtered, and concentrated. The crude product was
purified by radial chromatography (2 mm SiO2, 1-5% MeOH
in CH2Cl2) to yield the 1-N-methylindole tricycle as a white
solid: 44.9 mg (81%); mp 254-256 °C (dec); 1H NMR (300
MHz, DMSO-d6) δ 2.88 (m, 2H), 3.40 (m, 2H), 3.74 (s, 3H),
7.34 (t, J ) 7.7 Hz, 1H), 7.56 (m, 5H), 7.73 (d, J ) 7.7 Hz,
1H), 7.80 (d, J ) 7.7 Hz, 1H), 8.15 (br t, 1H). Anal. (C18H16N2O‚
0.75H2O) C, H, N.
1,5-N,N-Dim eth yl-2-p h en yl-3,4,5,6-tetr a h yd r o-1H-a ze-
p in o[5,4,3-cd ]in d ol-6-on e (Com p ou n d 30). A solution of
2-phenyl-3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-one (7,
0.91 mg, 0.35 mmol) in DMPU (2 mL) and THF (2 mL) was
cooled with an ice/water bath and treated with NaH (60%,
0.032 g, 0.80 mmol). The resulting yellow mixture was allowed
to stir at 0 °C for 30 min and then was treated with neat MeI
(0.88 mmol, 2.5 equiv). The mixture was allowed to warm to
room temperature and was stirred for 2 h. The reaction was
quenched with aqueous saturated NH4Cl and extracted with
EtOAc (3 × 10 mL). The organic solution was washed with
water and brine, dried (Na2SO4), filtered, and concentrated to
give the crude product as an oil. The crude product was
crystallized (THF/hexanes) to give the 1,5-N,N-dimethylindole
tricycle, 0.090 mg (89%), as an off-white solid: mp 175-177
°C; 1H NMR (300 MHz, DMSO-d6) δ 2.91 (m, 2H), 3.19 (s, 3H),
3.65 (m, 2H), 3.75 (s, 3H), 7.34 (t, J ) 7.8 Hz, 2H), 7.58 (m,
5H), 7.72 (d, J ) 7.8 Hz, 1H), 7.79 (d, J ) 7.8 Hz, 1H). Anal.
(C19H18N2O‚0.5H2O) C, H, N.
2-(4-(N,N-Dim et h yla m in o)m et h ylp h en yl)-3,4,5,6-t et -
r ah ydr o-1H-azepin o[5,4,3-cd]in dol-6-on e (Com pou n d 25).
The aldehyde 23 (310 mg, 1.07 mmol) in MeOH (40 mL) was
treated with dimethylamine (2 M solution in MeOH, 6.41
mmol). The solution was cooled with an ice/water bath a
treated dropwise with a solution of sodium cyanoborohydride
(74 mg, 1.18 mmol) and zinc chloride (80 mg, 0.59 mmol) in
MeOH (10 mL). The resulting solution was adjusted to pH 6-7
with 2 M methanolic HCl. After the mixture was stirred for
30 min, the reaction was quenched with concentrated HCl (0.2
mL) and the methanol was removed by evaporation. The
residue was diluted with water (30 mL). The solution was
adjusted to pH 10-11 with KOH(s) and extracted with CH2Cl2
(30 mL × 3). The organic solution was washed with water and
brine, dried (Na2SO4), filtered, and concentrated. The crude
product was crystallized (CH2Cl2/MeOH/hexanes) to give 2-(4-
(N,N-dimethylamino)methylphenyl)-3,4,5,6-tetrahydro-1H-
azepino[5,4,3-cd]indol-6-one, 245 mg (72%), as an off-white
solid: mp 226-229 °C (dec); 1H NMR (300 MHz, DMSO-d6) δ
2.18 (s, 6H), 3.06 (m, 2H), 3.40 (m, 2H), 3.44 (s, 2H), 7.21 (t, J
) 7.7 Hz, 1H), 7.43 (d of ABq, J ) 7.9 Hz, 2H), 7.56 (d, J )
7.7 Hz, 1H), 7.61 (d of ABq, J ) 7.9 Hz, 2H), 7.69 (d, J ) 7.7
Hz, 1H), 8.05 (br t, 1H), 11.53 (br s, 1H). HRMS (FAB, MH+)
calcd for
C20H22N3O: 320.1763. Found: 320.1753. Anal.
(C20H21N3O‚0.55H2O) C, H, N.
2-(4-P yr r olid in -1-ylm eth ylp h en yl)-3,4,5,6-tetr a h yd r o-
1H-a zep in o[5,4,3-cd ]in d ol-6-on e (Com p ou n d 26). As de-
scribed for compound 25, the para aldehyde 23 (150 mg, 0.52
mmol) in MeOH (10 mL) was treated with pyrrolidine (0.26
mL, 3.10 mmol) and a solution of sodium cyanoborohydride
(0.57 mmol) and zinc chloride (0.28 mmol) in MeOH (1.1 mL)
to give, after recrystallization (CH2Cl2/MeOH/hexanes), 2-(4-
pyrrolidin-1-ylmethylphenyl)-3,4,5,6-tetrahydro-1H-azepino-
[5,4,3-cd]indol-6-one, 141 mg (79%), as pale-yellow solid: mp
1
221-225 °C (dec); H NMR (300 MHz, DMSO-d6) δ 1.71 (m,
2-P h en yl-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]in d ole-6-
th ion e (Com p ou n d 31). Compound 7 (48.6 mg, 0.18 mmol)
in toluene (2 mL) was treated with Lawesson’s reagent (75
mg, 0.18 mmol) at room temperature. The solution was heated
at reflux for 2 h and then was allowed to cool to room
temperature and diluted with water. The mixture was ex-
tracted with EtOAc (3 × 5 mL). The organic solution was
washed with water and brine, dried (Na2SO4), filtered, and
concentrated. The crude product was crystallized (CH2Cl2/
hexanes) to give the thioamide, 34.4 mg (68%), as a yellow
solid: mp 223-226 °C (dec); 1H NMR (300 MHz, DMSO-d6) δ
3.10 (m, 2H), 3.50 (m, 2H), 7.23 (app t, J ) 7.8 Hz, 1H), 7.57
(m, 1H), 7.61 (m, 3H), 7.69 (m, 2H), 8.19 (d, J ) 7.6 Hz, 1H),
10.56 (br t, 1H), 11.68 (br s, 1H). HRMS (FAB, MH+) calcd for
4H), 2.46 (m, 4H), 3.06 (m, 2H), 3.41 (m, 2H), 3.63 (s, 2H),
7.21 (t, J ) 7.8 Hz, 1H), 7.45 (d of ABq, J ) 8.2 Hz, 2H), 7.55
(dd, J ) 7.9, 0.9 Hz, 1H), 7.59 (d of ABq, J ) 8.2 Hz, 2H), 7.68
(br d, 1H), 8.07 (br t, 1H), 11.54 (br s, 1H). HRMS (FAB, MH+)
calcd for
C22H24N3O: 346.1919. Found: 346.1911. Anal.
(C23H25N3O‚0.5H2O) C, H, N.
2-(3-Dim eth ylam in om eth ylph en yl)-3,4-dih ydr opyr r olo-
[4,3,2-d e]isoqu in olin -5-(1H)-on e (Com p ou n d 27). A pro-
cedure, starting with the meta aldehyde 20, and similar to
that described for compound 28 (vide infra) was used to
prepare the desired target in 42% yield: 1H NMR (300 MHz,
DMSO-d6) 2.18 (s, 6H), 3.45 (s, 2H), 5.03 (s, 2H), 7.20-7.30
(m, 2H), 7.35 (d, J ) 6.0 Hz, 1H), 7.40-7.58 (m, 3H), 7.60 (s,
1H), 7.79 (br s, 1H), 11.68 (br s, 1H); HRMS (M+) 306.1626.
C
17H15N2S: 279.0956. Found: 279.0952. Anal. (C17H14N2S‚
0.25H2O) C, H, N, S.
2-(4-Dim eth ylam in om eth ylph en yl)-3,4-dih ydr opyr r olo-
[4,3,2-d e]isoqu in olin -5-(1H)-on e (Com p ou n d 28). To a
solution of 2 M dimethylamine in MeOH (0.81 mL, 1.61 mmol)
was added 5 N HCl/MeOH (0.11 mL, 0.536 mmol) followed by
a suspension of the aldehyde 21 (0.074 g, 0.268 mmol) in 3
mL of MeOH and NaBH3CN (0.017 g, 0.268 mmol). The
resulting suspension was stirred for 72 h at room temperature.
Concentrated HCl was added until the pH was less than 2,
and the MeOH was removed in vacuo. The residue was taken
up in H2O and extracted with EtOAc. The aqueous solution
was brought to about pH 9 with solid KOH and extracted with
EtOAc. The organic layer was dried over MgSO4 and concen-
trated to give a yellow solid that was purified by flash silica
gel chromatography eluting with a gradient of 3% MeOH in
CHCl3 to 10% MeOH/NH3 in CHCl3 to give 0.023 g (28%) of
an orange solid: 1H NMR (300 MHz, DMSO-d6) 2.17 (s, 6H),
3.44 (s, 2H), 5.04 (s, 2H), 7.19 (t, J ) 6 Hz, 1H), 7.33 (d, J )
2-(4-Flu or oph en yl)-3,4,5,6-tetr ah ydr o-1H-azepin o[5,4,3-
cd ]in d ole (Com p ou n d 32). A solution of 2-(4-fluorophenyl)-
3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-one (13, 0.206
g, 0.74 mmol) in THF (10 mL) was treated with solid LAH
(95%, 0.140 g, 3.68 mmol). The mixture was heated at reflux
under argon for 6 h, resulting in a yellow mixture. The mixture
was allowed to cool to room temperature and then was cooled
further with an ice/water bath. Ice-cold 1 M aquoeus HCl (3
mL × 2) was added portionwise and alternating with ice-cold
water (3 mL). The mixture was extracted with CH2Cl2 (15 mL
× 3), washed with water and brine, dried (Na2SO4), and
concentrated. The crude product was recrystallized (CH2Cl2/
MeOH/hexanes) to give the azatricycle, 0.142 mg (72%), as a
bright, white solid: 1H NMR (300 MHz, DMSO-d6) δ 3.04 (m,
4H), 4.19 (s, 2H), 6.73 (app d, J ) 7.2 Hz, 1H), 6.99 (t, J ) 7.2
Hz, 1H), 7.19 (app d, J ) 7.2 Hz, 1H), 7.34 (m, 2H), 7.66 (m,

4972 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Canan Koch et al.
2H), 11.22 (s, 1H). HRMS (FAB, MH+) calcd for C17H16FN2:
267.1297. Found: 267.1298. Anal. (C17H15FN2) C, H, N.
1H); MS (electrospray, [M + Na]+) 266. Anal. (C13H13N3O2‚
0.4H2O) C, H, N.
6-Oxo-1,3,4,5-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ole-2-
ca r boxylic Acid Isop r op yla m id e (Com p ou n d 37). 6-Oxo-
3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indole-2-carboxylic acid
(34, 60 mg, 0.26 mmol), isopropylamine (17 mg, 0.29 mmol),
and diisoprophylethylamine (168 mg, 1.30 mmol) were dis-
solved in 5 mL of dry DMF. HATU (173 mg, 0.46 mmol) was
added, and the resulting mixture was stirred at room tem-
perature under argon for 3 days. The reaction mixture was
partitioned between water and 25% iPrOH/CHCl3. The layers
were separated, and the aqueous layer was extracted thrice
6-Oxo-3,4,5,6-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ole-2-
ca r boxylic Acid Meth yl Ester (Com p ou n d 33). 2-Iodo-
1,3,4,5-tetrahydroazepino[5,4,3-cd]indol-6-one (6, 85 mg, 0.28
mmol), palladium tetrakis(triphenylphosphine) (19 mg, 0.02
mmol), and triethylamine (52 mg, 0.51 mmol) were combined
in toluene/methanol (8:2 (v/v), 2 mL). Carbon monoxide gas
was bubbled through the mixture for 10 min. The reaction
mixture was then heated at 85 °C in a sealed tube for 16 h.
The solvent was evaporated, and the orange solid was purified
by radial chromatography (chloroform to 5% methanol in
chloroform). The white solid was recrystallized (chloroform/
methanol/hexanes) to yield 6-oxo-3,4,5,6-tetrahydro-1H-aze-
pino[5,4,3-cd]indole-2-carboxylic acid methyl ester, 39 mg
i
with 25% PrOH/CHCl3. The combined organic layers were
dried (MgSO4) and concentrated in vacuo, leaving an off-white
solid that was recrystallized from chloroform/methanol to yield
6-oxo-1,3,4,5-tetrahydro-1H-azepino[5,4,3-cd]indole-2-carbox-
ylic acid isopropylamide as a white solid: 20 mg (28%); mp
261-262 °C (dec); 1H NMR (300 MHz, DMSO-d6) δ 1.20 (d, J
) 6.6 Hz, 1H), 3.22 (m, 2H), 3.38 (m, 2H), 4.90 (m, 1H), 7.32
(app t, J ) 7.8 Hz, 1H), 7.59 (d, J ) 8.1 Hz, 1H), 7.71 (d, J )
7.2 Hz, 1H), 7.81 (d, J ) 7.5 Hz, 1H), 8.10 (br t, 1H), 11.53 (br
s, 1H); MS (electrospray, MH+) 272. Anal. (C15H17N3O2‚0.2H2O)
C, H, N.
1
(100%), as an off-white solid: mp 266-267 °C; H NMR (300
MHz, DMSO-d6) δ 3.25 (m, 2H), 3.43 (m, 2H), 3.89 (s, 3H),
7.38 (app t, J ) 7.8 Hz, 1H), 7.61 (dd, J ) 8.1, 0.9 Hz, 1H),
7.74 (dd, J ) 7.5, 0.9 Hz, 1H), 8.17 (br t, 1H), 11.93 (br s, 1H);
MS (FAB, MH+) 245. Anal. (C13H12N2O3) C, H, N.
6-Oxo-3,4,5,6-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ole-2-
ca r boxylic Acid (Com p ou n d 34). 6-Oxo-3,4,5,6-tetrahydro-
1H-azepino[5,4,3-cd]indole-2-carboxylic acid octyl ester (pre-
pared in a manner similar to that described for 33 (350 mg,
1.02 mmol)) and lithium hydroxide (122 mg, 5.11 mmol) were
dissolved in 10 mL of 2:1 methanol/water and stirred at room
temperature for 24 h. The reaction mixture was diluted with
water and then washed twice with dichloromethane. The
aqueous solution was acidified to ca. pH 2 with concentrated
HCl. The white precipitate was collected by filtration, washed
with water, and dried in vacuo to yield 6-oxo-3,4,5,6-tetra-
hydro-1H-azepino[5,4,3-cd]indole-2-carboxylic acid, 235 mg
6-Oxo-1,3,4,5-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ole-2-
ca r boxylic Acid P h en yla m id e (Com p ou n d 38). In a man-
ner similar to that described for compound 37, 6-oxo-3,4,5,6-
tetrahydro-1H-azepino[5,4,3-cd]indole-2-carboxylic acid (34, 60
mg, 0.26 mmol) was coupled to aniline (27 mg, 0.29 mmol) to
yield 6-oxo-1,3,4,5-tetrahydro-1H-azepino[5,4,3-cd]indole-2-
carboxylic acid phenylamide as a white solid: 55 mg (69%);
1
mp 320-322 °C (dec); H NMR (300 MHz, DMSO-d6) δ 3.28
(m, 2H), 3.42 (m, 2H), 7.11 (app t, J ) 7.5 Hz, 1H), 7.37 (m,
3H), 7.64 (d, J ) 8.1 Hz, 1H), 7.74 (m, 3H), 8.15 (br t, 1H),
9.98 (br s, 1H), 11.78 (br s, 1H); MS (electrospray, MH+) 306.
Anal. (C18H15N3O2‚0.25H2O) C, H, N.
1
(99%), as a white solid: mp 298-299 °C (dec); H NMR (300
MHz, DMSO-d6) δ 3.17 (m, 2H), 3.41 (m, 2H), 7.35 (t, J ) 7.8
Hz, 1H), 7.59 (d, J ) 8.1 Hz, 1H), 7.73 (d, J ) 7.5 Hz, 1H),
8.14 (br t, 1H), 11.77 (br s, 1H), 13.14 (br s, 1H); MS
(electrospray, MH+): 231. Anal. (C12H10N2O3‚1.0H2O) C, H, N.
2-P yr idin -3-yl-1,3,4,5-tetr ah ydr oazepin o[5,4,3-cd]in dol-
6-on e (Com p ou n d 39). In a manner similar to that described
for compound 7, the tricyclic bromide (5, 300 mg, 1.13 mmol)
and 3-pyridylboronic acid (153 mg, 1.24 mmol) were coupled
to yield 2-pyridin-3-yl-1,3,4,5-tetrahydroazepino[5,4,3-cd]indol-
6-one, 75 mg (25%), as a light-brown solid: mp 260.5-262.0
°C; 1H NMR (300 MHz, DMSO-d6) δ 3.07 (m, 2H), 3.40 (m,
2H), 7.25 (app t, J ) 7.8 Hz, 1H), 7.57 (m, 2H), 7.71 (dd, J )
7.5, 0.9 Hz, 1H), 8.05 (m, 1H), 8.12 (br t, 1H), 8.59 (m, 1H),
8.88 (m, 1H), 11.75 (br s, 1H); MS (FAB, MH+) 264. Anal.
(C16H13N3O‚0.2H2O) C, H, N.
6-Oxo-3,4,5,6-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ole-2-
ca r bon itr ile (Com p ou n d 35). In a manner similar to that
described by Anderson et al.,30 2-iodo-1,3,4,5-tetrahydroaze-
pino[5,4,3-cd]indol-6-one (6, 100 mg, 0.32 mmol), sodium
cyanide (31 mg, 0.64 mmol), palladium tetrakis(triphenylphos-
phine) (19 mg, 0.05 mmol), and copper(I) iodide were combined
in a Schlenk tube. The vessel was evacuated and refilled with
argon gas three times. Degassed propionitrile (2 mL) was
added, and the reaction mixture was stirred at 80 °C under
an argon atmosphere for 15 h. The reaction mixture was
partitioned between water and 25% iPrOH/CHCl3. The layers
were separated, and the aqueous layer was extracted thrice
2-Na p h th a len -1-yl-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]-
in d ol-6-on e (Com p ou n d 40). In a manner similar to that
described for compound 7, the tricyclic bromide (5, 300 mg,
1.13 mmol) and 1-naphthaleneboronic acid (214 mg, 1.24
mmol) were coupled to yield 2-naphthalen-1-yl-1,3,4,5-tetra-
hydroazepino[5,4,3-cd]indol-6-one, 70 mg (20%), as an off-white
i
with 25% PrOH/CHCl3. The combined organic layers were
dried (MgSO4) and concentrated in vacuo. The yellow solid was
recrystallized from CH2Cl2/MeOH/hexanes to yield 6-oxo-
3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indole-2-carbonitrile, 38
1
solid: mp 305 °C (dec); H NMR (300 MHz, DMSO-d6) δ 2.70
1
(m, 2H), 3.38 (m, 2H), 7.25 (app t, J ) 7.5 Hz, 1H), 7.61 (m,
5H), 7.75 (dd, J ) 7.5, 0.9 Hz, 1H), 7.82 (m, 1H), 8.06 (m, 3H),
11.67 (br s, 1H); MS (FAB, MH+) 313. Anal. (C21H16N2O‚
0.2H2O) C, H, N.
mg (56%), as a pale-yellow solid: mp 315 °C (dec); H NMR
(300 MHz, DMSO-d6) δ 3.04 (m, 2H), 3.47 (m, 2H), 7.46 (t, J
) 7.5 Hz, 1H), 7.64 (dd, J ) 8.1, 0.9 Hz, 1H), 7.81 (dd, J )
7.2, 0.9 Hz, 1H), 8.24 (br t, 1H), 12.44 (br s, 1H); MS
(electrospray, [M + Na]+) 234. Anal. (C12H9N3O) C, H, N.
2-Bip h en yl-3-yl-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]in -
d ol-6-on e (Com p ou n d 41). As described for compound 7, the
tricyclic bromide (5, 300 mg, 1.13 mmol) and biphenyl-3-
boronic acid (213 mg, 0.83 mmol) were coupled to yield
2-biphenyl-3-yl-1,3,4,5-tetrahydroazepino[5,4,3-cd]indol-6-
one, 116 mg (30%), as an off-white crystalline solid: mp 160-
163 °C; 1H NMR (300 MHz, DMSO-d6) δ 3.13 (m, 2H), 3.42
(m, 2H), 7.24 (app t, J ) 7.8 Hz, 1H), 7.42 (m, 1H), 7.61 (m,
7H), 7.79 (m, 2H), 7.94 (b s, 1H), 8.10 (br t, 1H), 11.67 (br s,
1H); MS (FAB, MH+) 339. Anal. (C23H18N2O) C, H, N.
2-Th iop h en -2-yl-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]in -
d ol-6-on e (Com p ou n d 42). In a manner similar to that
described for compound 7, the tricyclic bromide (5, 300 mg,
1.13 mmol) and thiophene-2-boronic acid (159 mg, 1.24 mmol)
were coupled to yield 2-thiophen-2-yl-1,3,4,5-tetrahydroaze-
pino[5,4,3-cd]indol-6-one, 171 mg (56%), as a beige solid: mp
220.5-222.5 °C; 1H NMR (300 MHz, DMSO-d6) δ 3.08 (m, 2H),
6-Oxo-3,4,5,6-tetr a h yd r o-1H-a zep in o[5,4,3-cd ]in d ole-2-
ca r boxylic Acid Meth yla m id e (Com p ou n d 36). 6-Oxo-
3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indole-2-carboxylic acid
methyl ester (33, 50 mg, 0.20 mmol) was suspended in 1 mL
of a 33% solution of methylamine in methanol. The suspension
was stirred at room temperature for 21 h. Another 2 mL of
33% methylamine in methanol was added, and the resulting
solution was stirred for another 8 h at room temperature and
then for 15 h at 30 °C. The reaction mixture was concentrated
in vacuo, leaving a yellow solid that was crystallized from
DMF/MeOH/CHCl3 to yield 6-oxo-3,4,5,6-tetrahydro-1H-aze-
pino[5,4,3-cd]indole-2-carboxylic acid methylamide, 36 mg
(72%), as a yellow solid: mp 321-322 °C (dec); 1H NMR (300
MHz, DMSO-d6) δ 2.81 (s, 3H), 3.15 (m, 2H), 3.40 (m, 2H),
7.32 (app t, J ) 7.8 Hz, 1H), 7.61 (d, J ) 8.1 Hz, 1H), 7.74 (d,
J ) 7.5 Hz, 1H), 7.95 (br q, 1H), 8.09 (br t, 1H), 11.46 (br s,

Poly(ADP-ribose) Polymerase-1 Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 4973
3.48 (m, 2H), 7.23 (m, 2H), 7.52 (m, 2H), 7.69 (m, 2H), 8.05
(br t, 1H), 11.60 (br s, 1H); MS (electrospray, MH+) 269. Anal.
(C15H12N2OS‚0.8H2O) C, H, N.
Refer en ces
(1) Ame, J . C.; Rolli, V.; Schreiber, V.; Niedergang, C.; Apiou, F.;
Decker, P.; Muller, S.; Hoger, T.; Menissier-De Murcia, J .; De
Murcia, G. PARP-2, a novel mammalian DNA damage-depend-
ent poly(ADP-ribose) polymerase. J . Biol. Chem. 1999, 274,
17860-17868.
(2) J ohansson, M. A human poly(ADP-ribose) polymerase gene
family (ADPRTL): cDNA cloning of two novel poly(ADP-ribose)
polymerase homologues. Genomics 1999, 57, 442-445.
(3) (a) J ean, L.; Risler, J . L.; Nagase, T.; Coulouarn, C.; Nomura,
N.; Salier, J . P. The nuclear protein PH5P of the inter-alpha-
inhibitor superfamily: a missing link between poly(ADP ribose)-
polymerase and the inter-alpha-inhibitor family and a novel
actor of DNA repair? FEBS Lett. 1999, 446, 6-8. (b) Kickhoefer,
V. A.; Siva, A. C.; Kedersha, N. L.; Inman, E. M.; Ruland, C.;
Streuli, M.; Rome, L. H. The 193-kD vault protein, VPARP, is a
novel poly(ADP-ribose) polymerase. J . Cell Biol. 1999, 146, 917-
928.
2-(1H-P yr r ol-2-yl)-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]-
in d ol-6-on e (Com p ou n d 43). In a manner similar to that
described for compound 7, the tricyclic bromide (5, 300 mg,
1.13 mmol) and 1-(t-butoxycarbonyl)pyrrole-2-boronic acid (263
mg, 1.24 mmol) were coupled with concomitant removal of the
BOC group to yield 2-(1H-pyrrol-2-yl)-1,3,4,5-tetrahydroaze-
pino[5,4,3-cd]indol-6-one, 81 mg (28%), as a greenish gray
solid: mp >400 °C (dec); 1H NMR (300 MHz, DMSO-d6) δ 3.02
(m, 2H), 3.42 (m, 2H), 6.22 (m, 1H), 6.44 (m, 1H), 6.97 (m,
1H), 7.14 (t, J ) 7.5 Hz, 1H), 7.49 (dd, J ) 8.1, 0.9 Hz, 1H),
7.64 (dd, J ) 7.5, 0.6 Hz, 1H), 7.98 (br t, 1H), 11.01 (br s, 1H),
11.13 (br s, 1H); MS (electrospray, MH+) 252. Anal. (C15H13N3O‚
0.4H2O) C, H, N.
2-(1H-In d ol-5-yl)-1,3,4,5-tetr a h yd r oa zep in o[5,4,3-cd ]in -
d ol-6-on e (Com p ou n d 44). In a manner similar to that
described for compound 7, the tricyclic bromide (5, 530 mg,
2.00 mmol) and indole-5-boronic acid (354 mg, 2.20 mmol) were
coupled to yield 2-(1H-indol-5-yl)-1,3,4,5-tetrahydroazepino-
[5,4,3-cd]indol-6-one, 396 mg (66%) as a beige solid: mp 315-
(4) Smith, S.; Giriat, I.; Schmitt, A.; De Lange, T. Tankyrase, a poly-
(ADP-ribose) polymerase at human telomeres. Science 1998, 282,
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(5) Smith, S. The world according to PARP. TIBS 2001, 26, 174-
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of the elongation and branching reaction of poly(ADP-ribose)-
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E. J . Mol. Biol. 1998, 278, 57-65. (b) Kameshita, I.; Matsuda,
Z.; Taniguchi, T.; Shizuta, Y. Poly (ADP-ribose) synthetase.
Separation and identification of three proteolytic fragments as
the substrate-binding domain, the DNA-binding domain, and the
automodification domain. J . Biol. Chem. 1984, 259, 4770-4776.
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(ADP-ribosyl)ation reactions in the regulation of nuclear func-
tions. Biochem. J . 1999, 342, 249. (b) De Murcia, G.; J osiane
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in base excision repair. Biochimie 1999, 81, 69-75. (b) Durkacz,
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Cancer Res. 1980, 40, 402. (f) Delaney, C. A.; Wang, L. Z.; Kyle,
S.; White, A. W.; Calvert, A. H.; Curtin, N. J .; Durkacz, B. W.;
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(adenosine diphosphoribose) polymerase inhibitors in a panel
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1
317 °C (dec); H NMR (300 MHz, DMSO-d6) δ 3.10 (m, 2H),
3.41 (m, 2H), 6.54 (m, 1H), 7.17 (t, J ) 7.8 Hz, 1H), 7.42 (m,
2H), 7.55 (m, 2H), 7.68 (d, J ) 7.5 Hz, 1H), 7.83 (br s, 1H),
8.05 (br t, 1H), 11.26 (br s, 1H), 11.48 (br s, 1H); MS
(electrospray, MH+) 302. Anal. (C19H15N3O‚0.25H2O) C, H, N.
Cr ysta llogr a p h y. Purified protein and cocrystals of the
C-terminal catalytic domain of chicken PARP were obtained
using procedures similar to those previously described.14 In
this study, 100 mM Tris, pH 8.5, 16-30% PEG-600, and 8%
isopropyl alcohol were used as the precipitant. X-ray diffraction
data were collected at -4 °C using monochromatized Cu Ka
radiation from a rotating anode generator and a MAR345
image plate. Data were processed with DENZO and
SCALEPACK.31 The crystal space group is P212121. For
compound 7, the cell constants are 58.98, 64.76, and 96.88 Å.
Data were measured from 20 to 2.3 Å in resolution; 74 743
reflections were scaled and merged into 16 519 unique reflec-
tions. The overall Rmerge is 7.3%, the ratio I/σ(I) is 19.1, and
the data are 96.7% complete. In the high-resolution shell
(2.38-2.30 Å), there are 1619 unique reflections, the overall
Rmerge is 27.8%, the ratio I/σ(I) is 4.1, and the data are 96.8%
complete. For compound 25, the cell constants are 59.13, 64.46,
and 97.47 Å. Data were measured from 20 to 2.3 Å in
resolution; 72 655 reflections were scaled and merged into
17 066 unique reflections. The overall Rmerge is 7.7%, the ratio
I/σ(I) is 14.3, and the data are 98.8% complete. In the high-
resolution shell (2.38-2.30 Å), there are 1679 unique reflec-
tions, the overall Rmerge is 30.0%, the ratio I/σ(I) is 3.4, and
the data are 98.8% complete.
These structures of PARP with bound inhibitor were solved
by difference maps using the data described above and a model
from an earlier structure,14c followed by refinement using the
computer program CNX2000.32 For compound 7, the conven-
tional and free R factors after refinement are 18.1% and 24.0%
for 13 485 and 1429 reflections, respectively. The non-hydrogen
model contains 2764 protein atoms, 20 inhibitor atoms, and
84 solvent atoms. The root-mean-square (rms) deviations
between ideal and model bond distances and angles are 0.006
Å and 1.23°. For compound 25, the conventional and free R
factors after refinement are 18.5% and 24.2% for 13 645 and
1468 reflections, respectively. The non-hydrogen model con-
tains 2764 protein atoms, 24 inhibitor atoms, and 86 solvent
atoms. The rms deviations between ideal and model bond
distances and angles are 0.006 Å and 1.23°.
Ack n ow led gm en t. We thank Gilbert de Murcia for
the baculovirus constructs used to produce hPARP-1-
FL and cPARP-1-CD for assays and crystallography and
also for providing us with protein purification protocols.
We thank Michelle Yang for assistance with manuscript
preparation.
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