Design and Synthesis of N-Substituted Indazole-3-Carboxamides as Poly(ADP-ribose)polymerase-1 (PARP-1) Inhibitors

Article abstract of DOI:10.1111/j.1747-0285.2011.01302.x

A group of novel N-1-substituted indazole-3-carboxamide derivatives were synthesized and evaluated as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). A structure-based design strategy was applied to a weakly active unsubstituted 1H-indazole-3-carboxamide 2, by introducing a three carbon linker between 1H-indazole-3-carboxamide and different heterocycles, and led to compounds 4 [1-(3-(piperidine-1-yl)propyl)-1H-indazole-3-carboxamide, IC₅₀=36μm] and 5 [1-(3-(2,3-dioxoindolin-1-yl)propyl)-1H-indazole-3-carboxamide, IC₅₀= 6.8μm]. Compound 5 was evaluated in rats for its protective action against diabetes induced by a treatment with streptozotocin, a known diabetogenic agent. In addition to preserving the ability of the pancreas to secrete insulin, compound 5 was also able to attenuate the ensuing hyperglycemic response to a significant extent.

Full text of DOI:10.1111/j.1747-0285.2011.01302.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01302.x
Chem Biol Drug Des 2012; 79: 488–496
Research Article
Design and Synthesis of N-Substituted Indazole-3-
Carboxamides as Poly(ADP-ribose)polymerase-1
(PARP-1) Inhibitorsꢀ
In spite of a lack of a complete understanding of the various path-
ways leading to PARP-1 activation, it has, however, become appar-
ent that the inhibition of PARP-1 with appropriate pharmacological
agents may translate into protection against the development of
diabetes (7–9). Numerous studies in laboratory animals, in which
diabetes was induced by using diabetogenic agents such as strepto-
zotocin (STZ) or alloxan (10–12), have suggested the possibility of
using PARP-1 inhibitors as a therapeutic approach for the preven-
tion of diabetes and its complications. For example, experimental
work in rodents with two known PARP-1 inhibitors, 3-aminobenza-
mide (3-AB) and nicotinamide (NI), has conclusively shown that the
administration of these compounds, concurrently with or shortly
after STZ, can prevent the diabetogenic effects of STZ in vivo, as
manifested by the attenuation of hyperglycemia and hypoinsulin-
emia and by the increase in the pancreatic synthesis and secretion
of insulin (7,8). Therefore, PARP-1 inhibitors may prove beneficial in
preventing or delaying the onset as well as the complications of
diabetes. The purpose of this article is to describe the synthesis,
structure–activity relationship (SAR) study, and in vitro and in vivo
evaluation of N-1-substituted indazole-3-carboxamide derivatives as
PARP-1 inhibitors and antidiabetogenic agents.
Maulik R. Patel, Kashyap G. Pandya, Cesar
A. Lau-Cam, Satyakam Singh, Maria A.
Pino, Blase Billack, Kurt Degenhardt and
Tanaji T. Talele*
Department of Pharmaceutical Sciences, College of Pharmacy and
Allied Health Professions, St. John's University, Queens, NY 11439,
USA
*Corresponding author: Tanaji Talele, talelet@stjohns.edu
ꢀPart of this work was presented at the 236th ACS National
Meeting, Philadelphia, PA, August 17-21, 2008 and 238th ACS
National Meeting, Washington DC, August 16–20, 2009.
A group of novel N-1-substituted indazole-3-carb-
oxamide derivatives were synthesized and evalu-
ated as inhibitors of poly(ADP-ribose)polymerase-1
(PARP-1). A structure-based design strategy was
applied to a weakly active unsubstituted 1H-indaz-
ole-3-carboxamide 2, by introducing a three car-
bon linker between 1H-indazole-3-carboxamide
and different heterocycles, and led to compounds
4
[1-(3-(piperidine-1-yl)propyl)-1H-indazole-3-carb-
oxamide, IC50 = 36 lM] and 5 [1-(3-(2,3-dioxoindo-
lin-1-yl)propyl)-1H-indazole-3-carboxamide, IC50
=
6.8 lM]. Compound 5 was evaluated in rats for its
protective action against diabetes induced by a
treatment with streptozotocin, a known diabeto-
genic agent. In addition to preserving the ability
of the pancreas to secrete insulin, compound 5
was also able to attenuate the ensuing hyperglyce-
mic response to a significant extent.
Molecular Design
Initial in vitro evaluation of 1H-indazole-3-carboxamide (2) as a
PARP-1 inhibitor indicated this compound to exhibit only a modest
(25% inhibition at 50 lM) activity. In the structure of 2, the N2
atom of the indazole core may lock the carboxamide group into the
desired conformation by an intramolecular hydrogen bond to form a
pseudotricyclic (6:5:5) ring system, whereas the R group may be
able to gain access to the adenosine (AD)-binding site of PARP-1
(Figure 1). Starting with compound 2 as a NI-mimic, we set out to
investigate the possibility of enhancing the PARP-1 inhibitory activ-
ity by structurally modifying this scaffold at the N-1 position by
altering heterocyclic ring at the terminal carbon atom of the propyl
linker. To attain this goal, a molecular docking study was carried
out using the X-ray crystal structure of the C-terminal catalytic frag-
ment of human PARP-1 (pdb ID: 2rcw) in complex with 2-substituted
benzimidazole-4-carboxamide inhibitor. Compound 2 was docked
into the catalytic site of human PARP-1 (Figure 2) using GLIDE DOCK-
ING program v5.0 (Schrodinger LLC, New York, NY, USA). The widely
accepted consensus pharmacophore for inhibition of PARP-1 (13),
developed by classical SAR studies and by modeling using the X-
ray crystal structure of the catalytic (NAD+-binding) domain (14,15),
is a primary or secondary benzamide, which makes important hydro-
Key words: 2,3-dioxoindoline, docking, diabetes, indazole-3-carbox-
amide, PARP-1
Received 21 May 2011, revised 3 December 2011 and accepted for pub-
lication 3 December 2011
Poly(ADP-ribose)polymerase-1 (PARP-1) is a chromatin-bound nuclear
enzyme involved in physiological functions such as DNA replication
and repair, cellular proliferation, differentiation, and apoptosis (1,2).
PARP-1 is a DNA nick sensor that by signaling the presence of
DNA damage facilitates DNA repair (3). In this role, this polymerase
catalyzes the addition of ADP-ribose units to histones and to vari-
ous DNA repair enzymes participating in replication, transcription,
differentiation, gene regulation, protein degradation, and spindle
maintenance (3–6).
488

Indazole-3-Carboxamides as PARP-1 Inhibitors
could accommodate heteroaryl alkyl chains amenable for the inves-
tigation of SAR. Using this line of reasoning, N-1-substituted deriva-
tives were synthesized and evaluated in vitro for inhibitory activity
toward PARP-1 and, more importantly, for singling out an analog
with a demonstrable high potency to prevent the development of
glucose intolerance in an animal model of diabetes.
Chemistry
Figure 1: Development of new class of indazole-3-carboxamide
poly(ADP-ribose)polymerase-1 inhibitors.
The synthesis of 1H-indazole-3-carboxamide and its derivatives is
depicted in Scheme 1. Compound 2 was obtained by treating com-
pound 1 with isobutyl chloroformate and N-methylmorpholine
(NMM) in THF followed by in situ addition of 30% aqueous ammo-
nia. Alkylation of compound 2 with 1-bromo-3-chloropropane under
basic condition provided compound 3. The selective alkylation of
the N-1 position remains a challenge, specifically for C3-unsubstitut-
ed indazoles (16). It has been previously reported that use of potas-
sium carbonate as a base yielded 92:1 ratio of N-1 versus N-2-
alkylated indazole derivatives (17). Moreover, the portion of N-2-
alkylated products is highly dependent on the bulkiness of the sub-
stituent at C3 position as exemplified through N-1 versus N-2 ratio
of 65:35 (when C3=-H) as opposed to the corresponding ratio of
95:5 (when C3=4-fluorophenyl) (18). On the basis of these prior find-
ings, we anticipated the formation of compound 3 (N-1 regioisomer)
as the major product. Compound 3 was assigned as N-1 regioisom-
er based on 13C NMR and 1D NOE spectral data. First, structural
assignment of compound 3 as the N-1 regioisomer was observed in
the 13C NMR spectra, where the resonance of C3 of the indazole
ring was downfield (d 140.7 ppm) and the resonance of C7a of the
indazole ring was upfield (d 137.5 ppm) in agreement with the pre-
viously reported values for N-1-substituted indazole derivatives (18–
20). Second evidence for the structural assignment of compound 3
was obtained by a 1D NOE experiment. Irradiation of the signal for
the 1¢-CH2-protons (triplet at d 4.59 ppm) resulted in a 4.7%
enhancement of the signal for the H7 proton (doublet at d
7.74 ppm) without any enhancement for the amide proton signals
(two singlet at d 7.69 and 7.40 ppm) (Supplementary data, Fig-
ure S1). These data clearly indicate that 1¢-CH2- and the C7-hydro-
gen are in close proximity to each other (Figure 3). Analysis of the
13C NMR and 1D NOE spectral data of compound 5 was found to
be in agreement with the aforementioned findings (Supplementary
data, Figure S2). Taken together, the 13C NMR and 1D NOE spectral
data described clearly support the structural assignment made. Tar-
get compound 4 was prepared by direct alkylation of compound 2
with commercially available 3-chloropropylpiperidine-1-yl. Target
compounds 5–10 were prepared by reacting compound 3 with the
corresponding heterocycles under basic condition.
Figure 2: Standard precision-Glide predicted binding pose for
compound 2 within the active site of poly(ADP-ribose)polymerase-1.
Important amino acids are depicted as sticks with the atoms col-
ored as carbon – green, hydrogen – white, nitrogen – blue, and
oxygen – red, whereas the inhibitor is shown as ball and stick
model with the same color scheme as above except carbon atoms
are represented in orange. Dotted red lines indicate hydrogen bond-
ing interaction.
gen bonds with Gly863 (-NH to Gly -C=O and -C=O to Gly -NH) and
Ser904 (-C=O to Ser -OH) in addition to p-stacking with Tyr907 and,
to some extent, with Tyr896 in the PARP-1 enzyme. In agreement
with the aforementioned findings, compound 2 bound to the NI site
by a sandwiched hydrophobic interaction with the phenyl rings of
Tyr896 and Tyr907 and the imidazole ring of His862. The amide
group formed a total of three hydrogen bonds represented by (-
C=O—HO- Ser904), (-C=O—HN- Gly863), and (-NH—O=C- Gly863).
On the basis of the molecular docking of compound 2, it was possi-
ble to verify that the N-1 position offered an open pocket that
Materials and Methods
Chemistry
Melting points (mp) were determined on a Thomas-Hoover capillary
melting point apparatus and are uncorrected. Reagents for organic
synthesis were purchased from Aldrich Chemical Co. (Milwaukee,
WI, USA), TCI America (Portland, OR, USA), Alfa Aesar (Ward Hill,
MA, USA), and Acros Organics (Antwerp, Belgium) and were used
Chem Biol Drug Des 2012; 79: 488–496
489

Patel et al.
Scheme 1: (a) i-BuOCOCl, NMM, THF, )20 ꢀC, 2 h; NH4OH (30%), rt, 1 h, 85%; (b) K2CO3, 3-chloropropylpiperidin-1-yl, CH3CN, reflux,
4 h, 75%; (c) K2CO3, 1-bromo-3-chloropropane, CH3CN, reflux, 4 h, 62%; (d) K2CO3, Heterocycle (Het), CH3CN, reflux, 4 h, 34–70%.
for C8H7N3O: C, 59.62; H, 4.38; N, 26.07. Found C, 59.43; H, 4.19;
N, 25.82.
N-(3-Chloropropyl)-1H-indazole-3-carboxamide
(3)
To a suspension of compound 2 (0.300 g, 1.86 mmol) in CH3CN
(10 mL), 1-bromo-3-chloropropane (0.321 g, 2.046 mmol) and anhy-
drous K2CO3 (0.564 g, 4.09 mmol) were added in succession, and
the mixture was stirred under reflux for 4 h. The mixture was then
filtered and concentrated under vacuum. The resulting solid was
purified by column chromatography (EtOAc:Hexane 60:40) to yield
the product as a white solid (0.286 g, 62%); m.p. 108–112 ꢀC; H
NMR (400 MHz, DMSO-d6, TMS) d 8.18 (d, 1H, J = 8.4 Hz), 7.74 (d,
1H, J = 8.0 Hz), 7.69 (s, 1H), 7.46 (t, 1H, J = 7.7 Hz), 7.40 (s, 1H),
7.26 (t, 1H, J = 7.5 Hz), 4.59 (t, 2H, J = 6.6 Hz), 3.65 (t, 2H,
J = 6.2 Hz), 2.33 (qn, 2H, J = 6.4 Hz); 13C NMR (100 MHz, DMSO-
d6, TMS) d 163.8, 140.7, 137.5, 126.6, 122.4, 122.3, 122.0, 110.1,
45.8, 42.4, 32.2.
Figure 3: The NOE detected for compound 3.
1
as received. All compounds were checked for homogeneity by TLC
1
using silica gel as a stationary phase. The H NMR spectra were
recorded on a Bruker 400 Avance DPX spectrometer outfitted with
a z-axis gradient probe. The chemical shifts are reported as parts
per million (d ppm) downfield from tetramethylsilane (TMS) as an
internal standard. Data are reported as follows: chemical shift, mul-
tiplicity (s) singlet, (d) doublet, (t) triplet, (qn) quintet, (m) multiplet,
(dd) doublet of doublet, and (bs) broad singlet. 1D-NOESY spectra
were processed with pulse width of 60 ms, power level of 64.7 dB,
phase correlation of 10.0, and 0.3 Hz line broadening. The C, H,
and N analyses were performed by Atlantic Microlabs, Inc., (Nor-
cross, GA, USA), and the observed values were within €0.4% of
calculated values.
1-(3-(Piperidin-1-yl)propyl)-1H-indazole-3-
carboxamide (4)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating compound 2 (0.150 g, 0.932 mmol) with N-
(3-chloropropyl)piperidine (0.164 g, 1.01 mmol) to yield compound 4
as a pale yellow solid (0.160 g, 59%); m.p. 92–94 ꢀC; 1H NMR
(400 MHz, DMSO-d6, TMS) d 8.16 (d, 1H, J = 8.4 Hz), 7.73 (d, 1H,
J = 8.4 Hz), 7.66 (s, 1H), 7.42 (t, 1H, J = 7.4 Hz), 7.37 (s, 1H), 7.23
(t, 1H, J = 7.4 Hz), 4.47 (t, 2H, J = 6.5 Hz), 2.15 (m, 6H), 2.00 (qn,
2H, J = 6.7 Hz), 1.44 (qn, 4H, J = 5.4 Hz), 1.34 (qn, 2H, J = 5.2 Hz);
13C NMR (100 MHz, DMSO-d6, TMS) d 164.0, 140.8, 137.2, 126.2,
122.2 (2C), 121.8, 110.4, 55.2, 53.9 (2C), 46.6, 26.6, 25.5 (2C), 24.1;
Anal. Calcd. for C16H22O1N4: C, 67.13; H, 7.69; N, 19.58. Found: C,
66.89; H, 7.75; N, 19.28.
1H-Indazole-3-carboxamide (2)
To a solution of indazole 3-carboxylic acid 1 (0.30 g, 1.85 mmol) in
anhydrous THF (7 mL) was added isobutyl chloroformate (0.28 g,
2.04 mmol) and NMM (0.21 g, 2.04 mmol) under a nitrogen atmo-
sphere and )20 ꢀC, and the mixture was stirred for 2 h. Then, to
this mixture, 5 mL of aqueous NH3 (30%) was added and the mix-
ture was stirred at rt for 1 h. The mixture was then diluted with
EtOAc (5 mL), partitioned with water (2 · 10 mL), dried over
Na2SO4, and concentrated in vacuum. Following purification of the
residue by column chromatography using CH2Cl2 ⁄ MeOH (95:5) as
the eluent, the product was obtained as white crystals (0.25 g,
85%); m.p. 284–286 ꢀC (lit. 285–286 ꢀC) (21); 1H NMR (400 MHz,
DMSO-d6, TMS) d 13.57 (s, 1H), 8.17 (d, 1H, J = 7.8 Hz), 7.76 (s,
1H), 7.60 (d, 1H, J = 8.4 Hz), 7.40 (t, 1H, J = 7.6 Hz), 7.36 (s, 1H),
7.23 (t, 1H, J = 7.2 Hz); 13C NMR (100 MHz, DMSO-d6, TMS) d
164.5, 141.2, 138.4, 126.5, 122.1, 121.8, 121.7, 110.7; Anal. Calc.
1-(3-(2,3-Dioxoindolin-1-yl)propyl)-1H-indazole-
3-carboxamide (5)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating 2,3-dioxoindoline (0.100 g, 0.680 mmol) with
the halide intermediate 3 (0.162 g, 0.680 mmol) to yield compound
5 as a brick-red solid (0.165 g, 70%); m.p. 186–188 ꢀC; 1H NMR
490
Chem Biol Drug Des 2012; 79: 488–496

Indazole-3-Carboxamides as PARP-1 Inhibitors
(400 MHz, DMSO-d6, TMS) d 8.19 (d, 1H, J = 8.6 Hz), 7.78 (d, 1H,
J = 8.6 Hz), 7.68 (s, 1H), 7.61 (t, 1H, J = 7.8 Hz), 7.54 (d, 1H,
J = 7.2 Hz), 7.44 (m, 2H), 7.27 (t, 1H, J = 7.2 Hz), 7.13 (m, 2H), 4.59
(t, 2H, J = 6.7 Hz), 3.76 (t, 2H, J = 6.7 Hz), 2.28 (qn, 2H,
J = 6.9 Hz); 13C NMR (100 MHz, DMSO-d6, TMS) d 183.3, 163.8,
158.2, 150.4, 139.6, 137.9, 137.3, 126.4, 124.3, 123.0, 122.3, 122.1,
121.9, 117.6, 110.5, 110.3, 46.2, 37.2, 26.9; Anal. Calcd. for
C19H16O3N4Æ … CH3OH: C, 66.37; H, 4.89; N, 16.09. Found: C, 66.15;
H, 4.73; N, 16.24.
C19H15O3N4BrÆ 1 ⁄ 3 EtOAc: C, 53.48; H, 3.90; N, 12.27. Found: C,
53.23; H, 3.68; N, 12.47.
1-(3-(5-Iodo-2,3-dioxoindolin-1-yl)propyl)-1H-
indazole-3-carboxamide (9)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating 5-iodo-2,3-dioxoindoline (0.150 g,
0.549 mmol) with halide intermediate 3 (0.130 g, 0.549 mmol) to
yield compound 9 as an orange solid (0.127 g, 49%); m.p. 212–
214 ꢀC; 1H NMR (400 MHz, DMSO-d6, TMS) d 8.15 (d, 1H,
J = 8.0 Hz), 7.90 (d, 1H, J = 8.6 Hz), 7.78 (s, 1H), 7.75 (d, 1H,
J = 8.0 Hz), 7.61 (s, 1H), 7.42 (m, 2H), 7.25 (t, 1H, J = 8.0 Hz), 7.00
(d, 1H, J = 7.6 Hz), 4.55 (t, 2H, J = 6.8 Hz), 3.75 (t, 2H, J = 7.4 Hz),
2.24 (qn, 2H, J = 6.6 Hz); 13C NMR (100 MHz, DMSO-d6, TMS) d
182.0, 163.8, 157.6, 149.7, 145.3, 140.6, 140.2, 137.3, 132.0, 126.5,
122.3, 122.2, 121.9, 119.7, 113.0, 110.3, 46.1, 37.3, 26.7; Anal.
Calcd. for C19H15O3N4IÆ 1 ⁄ 6 EtOAc: C, 48.31; H, 3.37; N, 11.46.
Found: C, 48.04; H, 3.68; N, 11.08.
1-(3-(5-Fluoro-2,3-dioxoindolin-1-yl)propyl)-1H-
indazole-3-carboxamide (6)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating 5-fluoro-2,3-dioxoindoline (0.100 g,
0.606 mmol) with halide intermediate 3 (0.144 g, 0.606 mmol) to
yield compound 6 as an orange-red solid (0.076 g, 34%); m.p. 184–
187 ꢀC; 1H NMR (400 MHz, DMSO-d6, TMS) d 8.15 (d, 1H,
J = 8.4 Hz), 7.75 (d, 1H, J = 8.4 Hz), 7.62 (s, 1H), 7.44 (m, 4H), 7.25
(t, 1H, J = 7.8 Hz), 7.17 (dd, 1H, J = 8.6 Hz, 5.0 Hz), 4.56 (t, 2H,
J = 6.8 Hz), 3.77 (t, 2H, J = 6.8 Hz), 2.25 (qn, 2H, J = 6.6 Hz); 13C
NMR (100 MHz, DMSO-d6, TMS) d 182.6, 163.8, 159.6, 157.2,
146.6, 140.6, 137.3, 126.4, 123.9, 123.6, 122.3, 122.1, 121.9, 118.6,
111.9, 110.3, 46.1, 37.3, 26.7; Anal. Calcd. for C19H15O3N4FÆ 1 ⁄ 3
EtOAc: C, 61.72; H, 4.50; N, 14.16. Found: C, 61.63; H, 4.35; N,
14.34.
1-(3-(5-Methoxy-2,3-dioxoindolin-1-yl)propyl)-
1H-indazole-3-carboxamide (10)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating 5-methoxy-2,3-dioxoindoline (0.100 g,
0.565 mmol) with halide intermediate 3 (0.134 g, 0.565 mmol) to
yield compound 10 as a dark brown solid (0.090 g, 42%); m.p.
161–163 ꢀC; 1H NMR (400 MHz, DMSO-d6, TMS) d 8.15 (d, 1H,
J = 8.6 Hz), 7.75 (d, 1H, J = 8.6 Hz), 7.63 (s, 1H), 7.42 (m, 2H), 7.24
(t, 1H, J = 7.6 Hz), 7.18 (dd, 1H, J = 8.4 Hz, 3.0 Hz), 7.11 (d, 1H,
J = 3.0 Hz), 7.07 (d, 1H, J = 8.0 Hz), 4.56 (t, 2H, J = 7.4 Hz), 3.75
(m, 5H), 2.24 (qn, 2H, J = 7.4 Hz); 13C NMR (100 MHz, DMSO-d6,
TMS) d 183.5, 163.8, 158.2, 155.6, 144.2, 140.6, 137.2, 126.4,
123.6, 122.3, 122.2, 121.9, 118.2, 111.6, 110.3, 109.2, 55.9, 46.2,
37.2, 26.9; Anal. Calcd. for C20H18O4N4: C, 63.49; H, 4.79; N, 14.81.
Found: C, 63.62; H, 4.79; N, 14.71.
1-(3-(5-Chloro-2,3-dioxoindolin-1-yl)propyl)-1H-
indazole-3-carboxamide (7)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating 5-chloro-2,3-dioxoindoline (0.100 g,
0.552 mmol) with halide intermediate 3 (0.131 g, 0.552 mmol) to
yield compound 7 as an orange solid (0.094 g, 44%); m.p. 195–
197 ꢀC; 1H NMR (400 MHz, DMSO-d6, TMS) d 8.15 (d, 1H,
J = 7.7 Hz), 7.75 (d, 1H, J = 8.6 Hz), 7.62 (m, 3H), 7.41 (m, 2H),
7.24 (t, 1H, J = 7.8 Hz), 7.18 (d, 1H, J = 8.2 Hz), 4.56 (t, 2H,
J = 7.2 Hz), 3.77 (t, 2H, J = 6.6 Hz), 2.25 (qn, 2H, J = 6.8 Hz); 13C
NMR (100 MHz, DMSO-d6, TMS) d 182.2, 163.8, 158.0, 148.9,
140.6, 137.3, 136.7, 127.3, 126.4, 123.8, 122.3, 122.1, 121.9,
119.1, 112.3, 110.4, 46.1, 37.3, 26.7; Anal. Calcd. for
C19H15O3N4Cl: C, 59.61; H, 3.95; N, 14.04. Found: C, 59.43; H,
3.71; N, 14.24.
PARP-1 inhibition assay
Inhibitory action of the various indazole-3-carboxamide derivatives
toward PARP-1 was determined using a commercially available mi-
croplate assay kit (Catalog # 4677-096-K; Universal Colorimetric
PARP Assay from Trevigen, Inc., Gaithersburg, MD, USA) and
according to the instructions provided by the manufacturer. Stock
solutions of the various test compounds were made in dimethyl
sulfoxide (DMSO), which were then serially diluted to the required
concentrations with distilled water. For the assay, each strip well
was filled with 10 lL of the inhibitor solution, 15 lL of diluted
PARP-1 enzyme (providing 0.5 Unit ⁄ well), and 25 lL of PARP Cock-
tail (consisting of biotinylated NAD, activated DNA in Tris–Cl pH
8.0, and EDTA). The strip wells were incubated at room temperature
for 60 min and then washed 4 times with phosphate-buffered sal-
ine (PBS: Na2HPO4, NaH2PO4, and NaCl) and 0.1% Triton. Then,
50 lL of diluted Strep-HRP (blocking solution) was added to each
well, and the strips were further incubated at room temperature for
60 min. After washing the wells 4 times each with PBS and 0.1%
Triton, they were mixed with 50 lL of TACS-Sapphireꢁ colorimetric
substrate and allowed to stand in the dark for 10-15 min. The
1-(3-(5-Bromo-2,3-dioxoindolin-1-yl)propyl)-1H-
indazole-3-carboxamide (8)
This compound was synthesized by the procedure used for com-
pound 3, by alkylating 5-bromo-2,3-dioxoindoline (0.100 g,
0.442 mmol) with halide intermediate compound
3 (0.105 g,
0.442 mmol) to yield compound 8 as a dark orange solid (0.092 g,
1
49%); m.p. 204–206 ꢀC; H NMR (400 MHz, DMSO-d6, TMS) d 8.15
(d, 1H, J = 8.0 Hz), 7.75 (m, 2H), 7.68 (d, 1H, J = 2.4 Hz), 7.62 (s,
1H), 7.42 (m, 2H), 7.25 (t, 1H, J = 7.4 Hz), 7.13 (d, 1H, J = 8.0 Hz),
4.56 (t, 2H, J = 7.0 Hz), 3.76 (t, 2H, J = 7.0 Hz), 2.24 (qn, 2H,
J = 6.8 Hz); 13C NMR (100 MHz, DMSO-d6, TMS) d 182.0, 163.8,
157.9, 149.3, 140.5, 139.5, 137.3, 126.6, 126.5, 122.3, 122.2, 121.9,
119.4, 114.8, 112.7, 110.4, 46.1, 37.3, 26.7; Anal. Calcd. for
Chem Biol Drug Des 2012; 79: 488–496
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Patel et al.
intensity of the blue color that developed in each well was read on
a microplate reader set at 630 nm. After stopping the reaction by
the addition of 50 lL of 5% phosphoric acid to each well, the
absorbance of the yellow color was measured at 450 nm. Parallel
experiments were conducted by substituting the test solution with
an equivalent volume of DMSO or distilled water to verify the
effect of the vehicles on the enzyme activity. All the samples were
tested in triplicate. Those compounds found to inhibit PARP-1 activ-
ity by ‡50% at a concentration of 50 lM were further tested using
various concentrations to obtain dose–response curves. To deter-
mine the IC50 value for each inhibitor, the average absorbance of
each inhibitor concentration was plotted against the log of the con-
centration of each respective inhibitor (semi-log plot) and the IC50
value for each plot was obtained using Regression Wizard from
SIGMA PLOT version 10.0 (Systat, San Jose, CA, USA). Data pre-
sented are the results of at least two independent experiments car-
ried out in triplicate. The results of these studies are presented as
mean € standard deviation (SD).
ethylene tubes. Plasma samples were obtained by centrifugation of
the blood samples at 5193 · g for 10 min, and decanting the clear
supernatants into clean 10-mL polyethylene tubes. The plasma sam-
ples were kept at 4 ꢀC in a refrigerator until needed for an assay.
Assays
Blood glucose was measured in a drop of tail vein blood using a
commercial glucometer (ACCU-CHEKꢂ Active and diabetes test
strips; Roche Diagnostics Corporation U.S., Indianapolis, IN, USA).
Plasma glucose was measured using a commercial enzymatic glu-
cose assay kit (Procedure No. 510, Sigma-Aldrich, St. Louis, MO).
Plasma insulin was measured using a commercially available insulin
ELISA immunoassay kit (Calbiotech, Inc., Spring Valley, CA, USA).
Statistical analysis
The results are expressed as the mean € SEM for n = 6 rats. Sta-
tistical differences from control groups were established by Stu-
dent's t-test, one-way analysis of variance, and Newman–Keuls
post hoc test. A difference was considered to be statistically signifi-
cant at p £ 0.05.
In vivo assessment of antidiabetic action
Animals
All the experiments were carried out on male Sprague-Dawley rats,
200–225 g in weight, obtained from Taconic Farms, Germantown,
NY, USA. The animals were housed in a room maintained at a tem-
perature of 21 € 3 ꢀC, a constant humidity, and a normal 12-h
light ⁄ 12-h dark cycle and allowed to acclimate to their new sur-
roundings for a period of 5 days before the start of an experiment.
During that time, the animals had free access to a commercial ani-
mal diet and filtered tap water. All experimental groups consisted
of six animals each and they were used in a non-fasted state. The
study received the approval of the Institutional Care and Use Com-
mittee of St. John's University, and the animals were cared in
accordance with the guidelines established by the United States
Department of Agriculture.
Molecular modeling
Molecular docking computations were carried out on a Dell Preci-
sion 470n workstation with the RHEL 4.0 operating system using
GLIDE 5.0 (Schrodinger, LLC). 3D Structures of compounds 2 and 5
were constructed using the fragment dictionary of MAESTRO 9.0
(Schrodinger, LLC), and geometry was optimized by Macromodel pro-
gram v9.5 using the OPLS-AA force field (22) with the steepest des-
cent followed by truncated Newton conjugate gradient protocol.
The X-ray crystal structure of PARP-1 in complex with 2-substituted
benzimidazole-4-carboxamide inhibitor (PDB ID: 2rcw), obtained from
the RCSB Protein Data Bank (PDB), was used in this study. The pro-
tein was optimized for docking using the 'Protein Preparation Wiz-
ard' and 'Prime-Refinement Utility' of MAESTRO 9.0.
Treatment solutions and dosing
Because the consensus pharmacophore for the inhibition of PARP-1
is a primary or secondary amide that makes important hydrogen
bonds with Gly863 and Ser904, the grid was centered manually
around the benzimidazole-4-carboxamide bound inhibitor; using the
amino group of amide moiety as hydrogen bond donor and back-
bone carbonyl oxygen atom of Gly863 as hydrogen bond acceptor to
guide the amide group of compounds 2 and 5 toward the backbone
of Gly863. Then, compounds 2 and 5 were docked into the PARP-1
active site with aforementioned constraints using the standard pre-
cision Glide docking method. The top scoring pose-PARP-1 complex
was then subjected to energy minimization using MACROMODEL pro-
gram v9.5 using the OPLS-AA force field (22) and used for graphical
analysis.
Solutions of the treatment compounds were prepared in either cit-
rate buffer pH 4.5 (STZ; 3-AB) or DMSO (compound 5) just prior to
an experiment. These solutions were administered by the intraperi-
toneal (i.p.) route, using a 2-mL plastic syringe fitted with a 25-
gauge needle, and in a volume not exceeding 2 mL. Control animals
received only physiological saline, citrate buffer pH 4.5, or DMSO in
a volume equal to that of a test compound. Diabetes was induced
with a single, 60 mg ⁄ kg, dose of STZ, and its occurrence was
ascertained by measuring the blood glucose twice, once in a drop
of tail vein blood at 24-hr post-STZ and once in a blood sample col-
lected following decapitation of the animals. In experiments in
which 3-AB or compound 5 were used, these agents were adminis-
tered by the i.p. route at a dose of 0.7, 1.4, and 2.1 mM ⁄ kg ⁄ 2 mL
at 45 min before a treatment with STZ.
Results and Discussion
Blood sample collections
The animals were killed by decapitation at 24-hr post-STZ, and their
blood samples were delivered to heparinized 10-mL stoppered poly-
The newly synthesized indazole-3-carboxamide derivatives were
tested in vitro for their PARP-1 inhibitory activity, results of which
are presented in Table 1. The benchmark PARP-1 inhibitors 3-AB
492
Chem Biol Drug Des 2012; 79: 488–496

Indazole-3-Carboxamides as PARP-1 Inhibitors
Table 1: Poly(ADP-ribose)polymerase-1 inhibition data for N-1
To establish the role of the phenyl portion of the 2,3-dioxoindoline
moiety in compound 5 toward PARP-1 inhibition, a short series of
5-substituted 2,3-dioxoindoline analogs were synthesized and sub-
jected to in vitro testing for PARP-1 inhibitory potency. From the
IC50 values shown in Table 1, it was possible to rank the PARP-1
inhibitory activity of the compounds in this series in the decreasing
order H>>F>Cl>>OMe>Br>>I. Thus, any substitution on the 2,3-dioxo-
indoline aryl ring appears to be detrimental to the PARP-1 inhibitory
action for reasons that are not clear at present. On the basis of the
aforementioned SAR data, indazole-3-carboxamide is a viable scaf-
fold for the synthesis of compounds with PARP-1 inhibitory activity;
however, the present results clearly suggest that its structural modi-
fication does not warrant that a desirable activity as a PARP-1
inhibitor will be realized. Therefore, our efforts were redirected to
identify alternate novel NI-mimicking scaffolds that unlike indazole-
3-carboxamide could be turned into highly active PARP-1 inhibitors.
substituted indazole-3-carboxamides
O
O
CONH2
NH
NH2
N
N
NH2
3-AB
Het
4-10
Compound
PJ-34
NHCOCH2NMe2
a
Het
IC50, lM
4
Piperidin-1-yl
2,3-dioxoindolin-1-yl
36.00 € 2.00
6.80 € 0.57
34.59 € 1.26
41.77 € 1.24
118.85 € 1.91
179.76 € 2.74
90.21 € 4.40
11.00
5
6
5-fluoro-2,3-dioxoindolin-1-yl
5-chloro-2,3-dioxoindolin-1-yl
5-bromo-2,3-dioxoindolin-1-yl
5-iodo-2,3-dioxoindolin-1-yl
5-methoxy-2,3-dioxoindolin-1-yl
7
8
9
10
3-AB
PJ-34
To be able to shed some light on the molecular interactions of
these inhibitors, we performed docking calculations on the most
potent compound 5 using the crystal structure of a 2-substituted
benzimidazole-4-carboxamide–PARP-1 complex (Figure 4). The hydro-
gen bonding pattern of the pharmacophoric amide group with
Gly863 and Ser904 was similar to that observed for compound 2.
The p-stacking interaction of the indazole moiety was also similar
to that of compound 2. The 2,3-dioxoindoline moiety binds into the
AD site as inferred from its location at van der Waals interacting
distances from the AD-binding residues such as Glu763, Asp766,
Asn767, and Asp770. The C2 carbonyl oxygen atom of the 2,3-dioxo-
indoline moiety forms a water (HOH502)-mediated hydrogen bond
with the backbone of Tyr896 (-C=O—H2O—HN- Tyr896) whereas
the C3 carbonyl oxygen atom was found to be positioned within
interacting distance (3.5 ꢀ) from HOH475. The phenyl portion of the
2,3-dioxoindoline is stabilized through pi-pi stacking interaction with
0.11
aIC50 values were determined by at least two independent experiments,
each carried out in triplicate.
(23,24) and PJ-34 ([2-(dimethylamino)-N-(6-oxo-5,6-dihydrophenan-
thridin-2-yl)acetamide]) (25) are included for comparison. Attachment
of a piperidine-1-propyl substituent at the N-1 position of compound
2 resulted in compound 4 (IC50 = 36 lM), a more potent inhibitor
of PARP-1 than compound 2. Analysis of the PARP-1 active site
suggested availability of a large pocket that could accommodate
bicyclic heterocycles capable of forming hydrogen bonding interac-
tions with active site residues and ⁄ or water molecules. To test this
concept, we have replaced the piperidine ring of compound 4 with
a
2,3-dioxoindoline moiety, which provided compound
5
(IC50 = 6.8 lM) with >5-fold improvement in potency relative to
compound 4.
Having established that the 2,3-dioxoindoline ring being optimum
for PARP-1 inhibitory activity when inserted at the terminal carbon
atom of the linker, we decided to examine the effect of introducing
a shorter (ethyl) and longer (butyl) linker on the PARP-1 inhibitory
activity of compound 5. To this end, 2,3-dioxoindoline analogs with
two (compound S-2) and four (compound S-4) carbon linkers
between the indazole-3-carboxamide and 2,3-dioxoindoline were
synthesized, tested, and proved to be significantly less active than
compound 5 (Supplementary data, Table S1). At this point, further
activity optimization efforts on 2,3-dioxoindoline derivatives were
directed at examining the effect of relocating the hydrogen bond
acceptor groups of the 2,3-dioxoindoline moiety of compound 5 as
well as of steric bulkiness. On this basis, a 1,3-dioxoisoindoline
analog (compound S-5) was synthesized and tested but found to
be significantly less potent than compound 5. Further removal of
the phenyl portion of the 1,3-dioxoisoindoline moiety resulted in
pyrrolidine-2,5-dione analog S-6, which also proved to be inferior
than compound 5. Next, we focused our attention to replace the
propyl linker with 2-propanol (compound S-8) and propanoyl (com-
pound S-10) linkers; however, both of these compounds showed 5-
to 6-fold less activity than compound 5 (Supplementary data,
Table S1).
Figure 4: Standard precision-Glide predicted binding pose for
compound 5 within the active site of poly(ADP-ribose)polymerase-1.
Inhibitor is shown in ball and stick model, while the active site
amino acids and water molecules are shown in stick model. Dotted
red lines indicate hydrogen bonding interaction, whereas dotted
black lines show the interacting distance. Color scheme is the same
as in Figure 2.
Chem Biol Drug Des 2012; 79: 488–496
493

Patel et al.
Tyr889. It is interesting to note that in the crystal structure of
PARP-1, the carboxylate group of Asp766 is located within interact-
ing distance (3.6 ꢀ) from the hydroxyl group of Tyr889 and this
interaction seem to have implications in preserving the secondary
structure and function of the enzyme (26,27). We speculate that the
2,3-dioxoindoline ring may be involved in disrupting the aforemen-
tioned interaction by occupying the space between the side chains
of Asp766 and Tyr889. The existence of no tolerance for substitu-
ents at the 5-position of the 2,3-dioxoindoline moiety may be the
result of steric hindrance with either the carboxylate group of
Asp766 or the hydroxyl group of Tyr889.
Table 2: The effects of 3-AB and compound 5 on the plasma
glucose and plasma insulin levels of rats made diabetic with strep-
tozotocin (STZ)a,b,c
Treatment
Plasma glucose, mg ⁄ dL Plasma insulin, lIU ⁄ mL
Physiological saline
Citrate buffer pH 4.5
DMSO
79.42 € 4.65
78.18 € 5.42
0.491 € 0.015
0.482 € 0.013
92.90 € 4.02
0.487 € 0.017
STZ
401.78 € 9.46***,ꢀꢀꢀ
87.38 € 1.76
0.113 € 0.007***,ꢀꢀꢀ
0.457 € 0.003
3-AB 2.1 mM ⁄ kg
5 1.4 mM ⁄ kg
98.05 € 3.38*,ꢀ
0.414 € 0.009
STZ + 3-AB 0.7 mM ⁄ kg 200.27 € 5.87***,ꢀꢀꢀ,ꢀꢀꢀ,
0.191 € 0.006***,ꢀꢀꢀ,ꢀꢀꢀ
STZ + 3-AB 1.4 mM ⁄ kg 170.69 € 11.68***,ꢀꢀꢀ,ꢀꢀꢀ 0.257 € 0.014**,ꢀꢀ,ꢀꢀꢀ
STZ + 3-AB 2.1 mM ⁄ kg 113.26 € 7.72**,ꢀꢀ,ꢀꢀꢀ
0.303 € 0.022*,ꢀ,ꢀꢀꢀ
171.60 € 4.20•••,***,ꢀꢀꢀ,# 0.206 € 0.007•••,ꢀꢀꢀ,ꢀꢀꢀ
99.91 € 6.00ꢀꢀꢀ,## 0.298 € 0.008••,ꢀꢀ,ꢀꢀꢀ,#
58.88 € 3.08••,ꢀꢀꢀ,ꢀꢀ,### 0.347 € 0.008•,ꢀ,ꢀꢀꢀ,#
Several studies have indicated that PARP-1 inhibitors participate in
the regeneration of pancreatic b-islet cells and, as a result, they
are able to delay or even reverse the progression of type 1 diabetes
mellitus and some of its complications (7,8,28). A pharmacological
agent that has been widely used to examine the beneficial effects
of PARP-1 inhibitors in animal models of experimental diabetes has
been STZ (11,12). STZ is a bacterial nitrosourea analog of D-glucose
(2-deoxy-2-[3-methyl-3-nitrosoureido-D]glucopyranose) that is prefer-
entially taken up by b-islet cells with the aid of the glucose trans-
porter GLUT2 (11). STZ induces diabetes by promoting DNA breaks
in insulin-producing pancreatic cells, mainly through alkylation and,
to a lesser extent, by the generation of reactive oxygen species and
reactive nitrogen species. These events will activate nuclear PARP-1
to synthesize large amounts of chromatin protein-bound ADP-ribose
polymer from NAD+ and for altering the chromosomal structure to
make the DNA-damaged site(s) accessible to repair enzymes (29).
However, overactivation of PARP-1 will ultimately lead to the mas-
sive utilization of NAD+, a precursor of ATP, within pancreatic islet
cells, and bring NAD+ and ATP contents to levels that are insuffi-
cient to meet the normal cellular energy requirements and are con-
ducive to decreased function and necrotic cell death. An alternative
view on the role of overactivation of PARP-1 as a determinant of
cell death is that it initiates a nuclear signal that propagates to the
mitochondrion and triggers the release of apoptosis-inducing factor.
Apoptosis-inducing factor then shuttles from the mitochondrion to
the nucleus, where it induces peripheral chromatin condensation,
large-scale fragmentation of DNA, and, ultimately, apoptotic b-cell
death (30). Irrespective of the mechanism of action of STZ in caus-
ing diabetes, this compound has been extensively utilized to induce
experimental diabetes in rodents, especially mice and rats, in stud-
ies aimed at demonstrating the in vivo effectiveness of PARP-1
inhibitors and other chemical agents to attenuate diabetic hypergly-
cemia and hypoinsulinemia. Using the rat as an experimental model,
it was possible to determine that an acute intraperitoneal (ip) treat-
ment with STZ (60 mg ⁄ kg in citrate buffer pH 4.5) led to a signifi-
cant increase in plasma glucose (by approximately 414%, p < 0.001
versus DMSO) and a significant decrease in plasma insulin (by 76%,
p < 0.001 versus DMSO) (Table 2). In contrast, an ip pretreatment
with compound 5, given at doses equal to 0.7 and 1.4 mM ⁄ kg, led
to a dramatic dose-related attenuation of the changes in plasma
glucose (increases equal to only 85% and 8%, respectively, when
compared to control animals receiving only DMSO, p < 0.001 versus
STZ), an effect that was accentuated further at a dose of
2.1 mM ⁄ kg (decrease of 37% below the control value, p < 0.001
versus STZ). On the other hand, a pretreatment with the same
doses of compound 5 counteracted the decrease in plasma insulin
STZ + 5 0.7 mM ⁄ kg
STZ + 5 1.4 mM ⁄ kg
STZ + 5 2.1 mM ⁄ kg
aValues for compound 5 were corrected for the contribution of the vehicle,
DMSO, as determined from values for animals on physiological saline.
bEach STZ + 3-AB treatment was compared with a STZ + 5 treatment at
the same dose.
cStatistical comparisons were vs. Citrate buffer pH 4.5 at *p < 0.05,
**p < 0.01, ***p < 0.001; vs. Physiological saline at ꢀp < 0.05, ꢀꢀp < 0.01,
ꢀꢀꢀp < 0.001; vs. STZ at ꢀꢀꢀp < 0.001; vs. DMSO at •p < 0.05, ••p < 0.01,
#
•••p < 0.001; vs. STZ + 3-AB at p < 0.05, ##p < 0.01, ###p < 0.001
induced by STZ in a dose-related manner (decreases ranging from
only 29–58%, p < 0.001 versus STZ). Moreover, a pretreatment with
identical doses of 3-AB, a prototypical PARP-1 inhibitor, led to a
weaker protective action against the hyperglycemia and hypoinsulin-
emia associated with STZ-induced diabetes than compound 5
(plasma glucose increases of 45-156%, plasma insulin decreases of
37-60% vs. normal (physiological saline) values, all differences at
p < 0.001 versus STZ). The return of the insulin level to ꢀ63% of
control with the highest dose of 3-AB tested is in line with the
value reported in the literature for this compound when given to
rats 15 min before STZ (31). Furthermore, the present results for
compounds 5 and 3-AB correlate well with their in vitro inhibitory
potencies on PARP-1 (IC50 = 6.80 lM for compound 5,
IC50 = 11.0 lM for 3-AB). Table 2 summarizes the results for the in
vivo evaluation of compounds 5 and 3-AB as antidiabetogenic
agents. Because PARP-1, and probably some of its isoforms, is
involved in the repair of damaged sites in DNA, it is likely that inhi-
bition of the enzyme will result into accumulation of dsDNA breaks
in normal tissues (13,32). Moreover, PARP inhibition may be associ-
ated with genomic instability because PARP has been described as
the 'guardian angel protecting the genome' (33). However, it may
also be noted that several PARP inhibitors are being advanced to
Phase II ⁄ III oncology clinical trials.
Conclusions
We have synthesized a series of novel N-1-substituted indazole-3-
carboxamide derivatives, demonstrating inhibitory activity toward
PARP-1. In this series, compound 5 (IC50 = 6.8 lM) was found to be
the most potent one. More importantly, compound 5 demonstrated
a potent dose-related antidiabetogenic activity in an STZ-based ani-
mal model of experimental diabetes because it helped to reduce
494
Chem Biol Drug Des 2012; 79: 488–496

Indazole-3-Carboxamides as PARP-1 Inhibitors
the hyperglycemia and hypoinsulinemia induced by STZ at a greater
extent than equivalent doses of 3-AB, a well-known PARP-1 inhibi-
tor. In a continuing search for PARP-1 inhibitors with inhibitory
potency greater than that of compound 5, further synthetic and
SAR work is presently ongoing and the results of which will be
communicated in future.
10. Charron M.J., Bonner-Weir S. (1999) Implicating PARP and NAD+
depletion in type I diabetes. Nat Med;5:269–270.
11. Schnedl W.J., Ferber S., Johnson J.H., Newgard C.B. (1994)
Streptozocin transport and cytotoxicity. Specific enhancement in
GLUT2-expressing cells. Diabetes;43:1326–1333.
12. Yamamoto H., Uchigata Y., Okamoto H. (1981) Streptozocin and
alloxan induce DNA strand breaks and poly (ADP-ribose) synthe-
tase in pancreatic islets. Nature;294:284–286.
Acknowledgments
13. Woon E.C.Y., Threadgill M.D. (2005) Poly(ADP-ribose)polymerase
inhibition – where now? Curr Med Chem;12:2373–2392.
14. Ruf A., Mennissier D.M., de Murcia G., Schulz G.E. (1996) Struc-
ture of the catalytic fragment of poly(AD-ribose) polymerase
from chicken. Proc Natl Acad Sci USA;93:7481–7485.
15. Ruf A., Mennissier D.M., de Murcia G., Schulz G.E. (1998) Inhibi-
tor and NAD+ binding to poly(ADP-ribose) polymerase as derived
from crystal structures and homology modeling. Biochemis-
try;37:3893–3900.
Support to TT in the form of start-up funds and resources from
the College of Pharmacy and Allied Health Professions, St.
John's University, Queens, NY, are gratefully acknowledged. We
thank Dr. Santosh Khedkar from Harvard Medical School and Dr.
V. Hariprasad from Huntsman Cancer Institute for many helpful
suggestions.
16. Saenz J., Mitchell M., Bahmanyar S., Stankovic N., Perry M.,
Craig-Woods B., Kline B., Yu S., Albizati K. (2007) Process develop-
ment and scale-up of AG035029. Org Process Res Dev;11:30–38.
17. Hunt K.W., Moreno D.A., Suiter N., Clark C.T., Kim G. (2009)
Selective synthesis of 1-functionalized-alkyl-1H-indazoles. Org
Lett;11:5054–5057.
Conflict of Interest
The authors declare no competing interests.
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Appendix S2. Preparation of 1-(2-(2,3-dioxoindolin-1-yl)ethyl)-1H-
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Appendix S3. Preparation of N-(4-chlorobutyl)-2,3-dioxoindoline.
Appendix S4. Preparation of 1-(4-(2,3-dioxoindolin-1-yl)butyl)-1H-
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1H-indazole-3-carboxamide.
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Appendix S6. Preparation of 1-(3-(2,5-dioxopyrrolidin-1-yl)propyl)-
1H-indazole-3-carboxamide.
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Appendix S7. Preparation of 1-(oxiran-2-ylmethyl)-1H-indazole-3-
carboxamide.
Appendix S8. Preparation of 1-(3-(2,3-dioxoindolin-1-yl)-2-hydro-
xypropyl)-1H-indazole-3-carboxamide.
Appendix S9. Preparation of 1-(3-chloropropanoyl)-1H-indazole-3-
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carboxamide.
Appendix S10. Preparation of 1-(3-(2,3-dioxoindolin-1-yl)propa-
noyl)-1H-indazole-3-carboxamide.
Supporting Information
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Any queries (other than missing material) should be directed to the
corresponding author for the article.
Additional Supporting Information may be found in the online ver-
sion of this article:
Figure S1. NOESY spectra for compound 3.
Figure S2. NOESY spectra for compound 5.
Table S1. PARP-1 inhibition data of N-1 substituted indazole-3-
carboxamides.
Appendix S1. Preparation of N-(2-chloroethyl)-2,3-dioxoindoline.
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