7-Azaindole-1-carboxamides as a new class of PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.12.031

7-Azaindole-1-carboxamides were designed as a new class of PARP-1 inhibitors The compounds displayed a variable pattern of target inhibition profile that, in part, paralleled the antiproliferative activity in cell lines characterized by homologous recombination defects A selected compound (1l; ST7710AA1) showed significant in vitro target inhibition and capability to substantially bypass the multidrug resistance mediated by Pgp In antitumor activity studies against the MX1 human breast carcinoma growth in nude mice, the compound exhibited an effect similar to that of Olaparib in terms of tumor volume inhibition when used at a lower dose than the reference compound Treatment was well tolerated, as no deaths or significant weight losses were observed among the treated animals

Full text of DOI:10.1016/j.bmc.2013.12.031

Bioorganic & Medicinal Chemistry 22 (2014) 1089–1103
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
7-Azaindole-1-carboxamides as a new class of PARP-1 inhibitors
Raffaella Cincinelli a, , Loana Musso a, , Lucio Merlini a, Giuseppe Giannini b, Loredana Vesci b,
Ferdinando M. Milazzo b, Nives Carenini c, Paola Perego c, Sergio Penco b, Roberto Artali d, Franco Zunino c,
Claudio Pisano b, Sabrina Dallavalle a,
⇑
a Department of Food, Environmental and Nutritional Sciences, Division of Chemistry and Molecular Biology, Università di Milano, via Celoria 2, 20133 Milano, Italy
b R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A.,via Pontina Km 30, 400, I-00040 Pomezia (RM), Italy
c Molecular Pharmacology Unit, Dept. Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, via Amadeo 42, I-20133 Milan, Italy
d Scientia Advice, di Roberto Artali, 20832 Desio (MB), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
7-Azaindole-1-carboxamides were designed as a new class of PARP-1 inhibitors. The compounds dis-
played a variable pattern of target inhibition profile that, in part, paralleled the antiproliferative activity
in cell lines characterized by homologous recombination defects. A selected compound (1l; ST7710AA1)
showed significant in vitro target inhibition and capability to substantially bypass the multidrug resis-
tance mediated by Pgp. In antitumor activity studies against the MX1 human breast carcinoma growth
in nude mice, the compound exhibited an effect similar to that of Olaparib in terms of tumor volume inhi-
bition when used at a lower dose than the reference compound. Treatment was well tolerated, as no
deaths or significant weight losses were observed among the treated animals.
Received 25 October 2013
Revised 12 December 2013
Accepted 16 December 2013
Available online 22 December 2013
Keywords:
PARP inhibitors
Synthesis
Molecular modelling
7-Azaindoles
Antitumor
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and radiation treatment,2,4 and clinical trials of combinations with
DNA-damaging agents are in progress.4b
Members of the Poly(ADP-ribose) polymerase (PARP) proteins
family are promising therapeutic targets which are involved in
maintaining DNA integrity and in regulation of programmed cell
death.1,2 So far 18 members have been described, PARP-1 and
PARP-2 being the best characterized ones. PARP-1, which is selec-
tively activated by DNA damage, catalyzes the transfer of ADP-
ribose units to a variety of nuclear proteins (histones, transcription
factors and PARP itself) using nicotinamide adenine dinucleotide
(NAD+) as a substrate, thereby leading to the formation of long
and branched poly(ADPribose)(PAR) chains.3 This is a key process
for the repair of DNA damage through the base excision repair
(BER) pathway.3
Given the biological role of the enzyme, the blockade of PARP-1
activity has the potential to prevent DNA damage repair leading
consequently to cell death. Since the efficacy of many anticancer
drugs implicating DNA damage is undermined by the emergence
of resistance, often due to enhanced DNA repair, PARP-1 inhibitors
have also potential therapeutic applications as chemo-sensitizers.
PARP inhibitors have been reported to sensitize cancer cells to drug
Moreover, cells carrying mutations in BRCA1/2 tumor suppres-
sor genes are sensitive to PARP inhibitors per se owing to their de-
fect in homologous recombination.5,6 Thus, PARP inhibitors are
promising agents as monotherapy in patients with mutated BRCA
genes.7 In this context, PARP inhibition results in synthetic lethal-
ity of cells characterized by homologous recombination defects.8
In addition to cancer, PARP-1 has been shown to be involved in
other disease conditions including inflammation,9 neuronal death
and ischemia.10
In the last three decades, a large number of PARP-1 inhibitors
have been reported,11,12 including Veliparib, 3AB, Olaparib, Ruca-
parib and Niraparib (Fig 1). Some of them are in advanced clinical
trials as single agents or in combination with DNA-damaging
drugs.4b,13,14
Most of the PARP inhibitors developed to date are structural
analogues of nicotinamide, and are thought to compete with
NAD itself at the level of the catalytic domain.
The human PARP proteins share only between 18% and 45% se-
quence homology in their catalytic domains, but crystal data indi-
cate that their structure is conserved and the mode of NAD+
cofactor binding is rather similar. Indeed, X-ray crystal struc-
tures11,12 and molecular modelling studies15–18 indicated that the
amide of nicotinamide makes three key hydrogen bonds with the
hydroxyl group of a serine residue and the amide backbone of a
⇑
Corresponding author. Tel.: +39 02 50316818; fax: +39 02 50316801.
E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
These authors contributed equally to the study.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.031

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R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
CONH2
CONH2
N
O
N
H
N
H
NH2
NH
3AB
ABT 888 (Veliparib)
N
O
F
H
N
N
O
N
KU-59436/AZD 2281 (Olaparib)
O
N
N
H
H
F
CONH2
AG-014699/ PF-01367338 (Rucaparib)
NH
N
N
MK4827 (Niraparib)
Figure 1. Structure of PARP inhibitors.
glycine residue, and that there is a stacking interaction with a con-
served tyrosine residue. All the available structures of PARP inhib-
itor complexes show compound bound to the pocket which retains
this moiety after NAD+ cleavage. One of the simplest compounds,
3-AB (Fig. 1), resembles nicotinamide and has been cocrystallized
with several PARP proteins. Extension of the compounds into the
donor site and toward the acceptor loops was used to improve
affinity and isoform selectivity. The success of this concept is illus-
trated considering the structure of the complex with ABT-888.19,20
The synthesis of conformationally constrained cyclic derivatives
of benzamide has demonstrated the crucial role of an anti-disposi-
tion of the amide bond. Thus, attempts to improve the affinity of
PARP inhibitors to the binding site have been made by locking
the carboxamide group, which is usually free to rotate, into the de-
sired anti-conformation (Fig. 2).11
optimal binding orientation was supported by molecular model-
ling studies (see later). On the basis of these studies, a number of
derivatives substituted in position 6 with aromatic or heterocyclic
groups were designed and synthesized.
To the best of our knowledge, the 7-azaindole structure repre-
sents a new scaffold for PARP inhibitors.
2. Results and discussion
2.1. Chemistry
Unsubstituted 7-azaindole-1-carboxamide (1a) was obtained
by reacting 7-azaindole with triphosgene, followed by NH3 treat-
ment in CH2Cl2 (Scheme 1).
The 6-substituted derivatives were synthesized by regioselec-
tive substitution21 of 7-azaindole N-oxide (3), in turn obtained by
oxidation of 7-azaindole with MCBA. Treatment of 3 with benzoyl
bromide in anhydrous toluene in the presence of hexamethyldisi-
lazane (HMDS) gave 4 in 71% yield. The acyl group on N-1 atom
was readily removed by basic treatment to give 6-bromo-7-azain-
dole 5 in 90% yield. Coupling with appropriate boronic acids via Pd/
catalyzed Suzuki reactions allowed to introduce different aromatic
and heteroaromatic rings at position 6. Finally, the carboxamide
group was installed by treatment with triphosgene, followed by
NH3.22 The yields of this last step were generally quite low
(Scheme 1).
Given the low solubility of the prototype 1a, further efforts con-
centrated on the addition of polar groups such as basic amines in
order to improve the solubility of these lipophilic compounds.
Unexpectedly, the reaction with triphosgene/NH3 on derivatives
bearing at the distal para-position a morpholinomethyl or a pipe-
ridinomethyl group failed.
In the present work, new 6-substituted pyrrolo[2,3-b]pyridine-
1-carboxamides (7-azaindole-1-carboxamides) (1) were designed.
These compounds incorporate a nitrogen atom into the aromatic
ring to lock the N-carboxamide group within a six-membered
‘pseudo-cycle’ through an intramolecular hydrogen bond. The
hypothesis that this interaction constrains the group into the
O
Ser904
Gly863
H
N
N
O
H
H
N
N
R
1
On the basis of these results, an exploration of alternative meth-
ods for the introduction of the carboxamide group was undertaken.
Azaindoles 1g, h, j, k, were obtained by treatment of compounds 6g,
h, j, k with 4-nitrophenylchloroformate to give the corresponding
Figure 2. Scheme of the locked conformation of a PARP inhibitor in the nicotin-
amide binding pocket and structure of pyrrolo[2,3-b]pyridine-1-carboxamides (7-
azaindoles) 1.

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
1091
c
a
b
N
H
N
N
H
N
H
N
Br
N
Br
N
N
O
59%
2
71%
5
4
90%
3
COPh
e
d
g
f
N
N
F
R
R
N
N
O
R
N
H
CONH2
57-89%
6b-l
17-85%
1a-l
7g,h, j, k
O
e
pNO2Ph
O
O
F
N
6b R = Ph
6c R = 4-(OMe)Ph
1a R = H
1b R = Ph
1h R =
6h R =
6i R =
N
N
N
H
N
Boc
N
O
O
1i R =
O
O
6d R = 4-(Me)2NPh
6e R = 4-FPh
1c R = 4-(OMe)Ph
1d R = 4-(Me)2NPh
1e R = 4-FPh
N
N
1j R =
1k R =
1l R =
6j R =
6k R =
6l R =
N
N
N
N
O
O
6f R = 4-Ph-Ph
6g R = 2-FPh
1f R = 4-Ph-Ph
1g R = 2-FPh
Scheme 1. Reagents and conditions: (a) MCPBA, K2CO3, H2O, rt; (b) PhCOBr, HMDS, toluene, N2, rt, 1 h; (c) NaOH 1 N, MeOH, rt, overnight; (d) RB(OH)2, PdCl2(dppf)CH2Cl2,
DME/H2O 3.5:1, NaHCO3, 110–115 °C; for 6f: PdCl2(dppf)CH2Cl2, 4-Br-biphenyl, bispinacolate diboron, KOAc, dioxane, 100 °C, 4 h, then 5, PdCl2(dppf)CH2Cl2, 2 M Na2CO3,
100 °C, 4 h; for 6h: 3-carboxy-4-fluorophenylboronic acid, PdCl2(dppf)CH2Cl2, DME/H2O 3.5:1, NaHCO3, 110–115 °C, then WSC, HOBt, DIPEA, 1-cyclopropylcarbonylpip-
erazine, DMF N2, rt, 20 h; for 6i: RB(OH)2, K2CO3, Pd(Ph3)4, DME/H2O 4:1, N2, 100 °C, 3 h; (e) triphosgene, DIPEA, toluene, 80 °C, then NH3; for 1a: 2, triphosgene, DIPEA, DCM,
0 °C, then NH3; for 1i: triphosgene, DIPEA, toluene, 80 °C, then NH3, then MeOH/HCl, reflux, 5 h; for 1l: LiHMDS, 4-nitrophenylchloroformate, THF, À78 °C, then NH4Cl; (f)
4-nitrophenylchloroformate, Bu4NBr, NaOH, toluene or DCM; (g) (NH4)2CO3, DMF, 40 °C.
4-nitrophenylcarbamates.23 The addition of ammonium carbonate
gave the desired carboxamides. Unfortunately, when the same
pathway was followed to obtain the aminoderivative 1l the yield
dropped to 17%.
Another synthetic route was then devised, in which the two fi-
nal steps, that is Suzuki coupling and urea formation, were re-
versed. 7-Azaindole-1-carboxamide was obtained in overall
quantitative yield by treatment of 5 with 4-nitrophenylchlorofor-
mate, followed by ammonium carbonate. Suzuki coupling with
two different amino-containing boronic acids gave the desired
compounds 1m, 1n together with consistent amounts of undesired
hydrolysis derivatives, whose separation by flash chromatography
resulted quite troublesome (Scheme 2).
initially obtained in very low yield, the synthetic strategy was re-
vised in the light of the various attempts made to install the trou-
blesome N-carboxamide group. After extensive experimentation,
the compound was prepared by the sequence reported in Scheme 5.
Protection of the carboxamide with one PMB group was accom-
plished reacting p-methoxybenzylamine with isopropenyl chloro-
formate to give the corresponding isopropenyl carbamate 17.26
This procedure showed to be very advantageous because it affor-
ded urea 18 in high yield in a single step from 6-bromoazaindole.
Moreover, the use of isopropenyl chloroformate instead of 4-nitro-
phenylchloroformate avoided the formation of 4-nitrophenol as a
side product and gave a crude that was easily purified, as the ace-
tone formed was removed by evaporation. Suzuki coupling, fol-
lowed by deprotection in TFA at reflux gave 1l in good yield. This
procedure affording the compound on a multi-gram scale can be
considered a convenient and rapid method to synthesize 6-substi-
tuted pyrrolo[2,3-b]pyridine-1-carboxamides (7-azaindole-1-car-
boxamides) in high yield and high purity.
In an attempt to overcome this problem, the carboxamide was
protected as PMB24 before Suzuki coupling and the protecting
group was removed in the last step. The sequence was followed
to synthesize compound 1o, whose preparation by the previously
reported methods failed (Scheme 3).
In an effort to evaluate the role of the urea group, a methylene
spacer was introduced between the azaindole core and the carbox-
amide group. Treatment of compound 5 with iodoacetamide and
final Suzuki coupling gave compound 11, in 25% overall yield
(Scheme 2).
To synthesize derivatives with a heterocyclic amine at position
6, 7-azaindole 2 was reacted with MCPBA followed by dimethyl
sulfate in acetonitrile and suitable amines to give the derivatives
6p–r.25 Treatment with triphosgene or 4-nitrophenylchlorofor-
mate, followed by quenching with ammonium carbonate, afforded
the corresponding carboxamides 1p–r (Scheme 4).
2.2. PARP-1 inhibitory activity
The compounds prepared in this study were evaluated for their
inhibitory activity against recombinant human PARP-1, expressed
as GST fusion protein, by means of a highly sensitive fluorescent
enzymatic assay. The results are summarized in Table 1. Three
PARP inhibitors–Olaparib,27ABT-88819 and MK-42728–were in-
cluded for comparative purposes.
The novel scaffold prototype 1a showed an encouraging activity
as PARP-1 inhibitor, displaying an IC50 value of 2.5 lM. Therefore, a
On the basis of biological activity results (see later) derivative 1l
was selected for further in vitro and in vivo investigation. As it was
more detailed exploration of the 7-azaindole-1-carboxamide
scaffold was undertaken. In an effort to improve the activity,

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R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
b
a
N
N
N
H
Br
N
Br
N
Br
N
CONH2
O
9
8
5
O
pNO2Ph
100%
(2 steps)
d
c
N
Br
N
N
CONH2
89%
N
10
CONH2
e
R
1m R =
1n R =
46%
29%
N
N
N
N
CONH2
N
N
11
29%
Scheme 2. Reagents and conditions: (a) 4-nitrophenylchloroformate, Bu4NBr, NaOH, DCM; (b) (NH4)2CO3, DMF, 40 °C; (c) RB(OH)2, PdCl2(dppf)CH2Cl2, DME/H2O 3.5:1,
NaHCO3, 80–110 °C; (d) NaH, DMF, 0 °C, iodoacetamide; (e) PdCl2(dppf)CH2Cl2, DME/H2O 3.5:1, NaHCO3, 110 °C.
CHO
b
a
N
H
N
H3CO
Br
N
H3CO
OCH3
13
N (PMB)2
12
14
71%
91%
O
d
c
N
N
N
N
N (PMB)2
NH2
O
O
1o
15
78%
57%
N
N
Scheme 3. Reagents and conditions: (a) 4-methoxybenzylamine, EtOH, reflux, 9 h, then NaBH4, MeOH; (b) 8, anhydrous DMF, 60 °C, 6 h; (c) pinacol 4[(N,N-
dimethylamino)methyl]phenylboronateÁHCl, PdCl2(dppf)CH2Cl2, NaHCO3, DME/H2O; 100 °C, 3 h; (d) TFA, reflux, 9 h.
a, b
c
N
H
N
H
N
N
R
N
R
N
CONH2
6p-r
2
29-56%
62-80%
d
e
N
N
1p R =
1q R =
1r R =
N
NO2
N
N
R
N
O
O
7q-r
Scheme 4. Reagents and conditions: (a) MCPBA, DME, rt; (b) Me2SO4, base, 50–55 °C, MeCN; (c) triphosgene, DIPEA, toluene, 80 °C, then NH3; (d) 4-nitrophenylchloro-
formate, Bu4NBr, NaOH, toluene, reflux; (e) (NH4)2CO3, DMF, 45 °C.

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
1093
O
a
b
NH2
N
O
H
H3CO
H3CO
17
16
N
N
c
N
Br
N
CO
N
CO
19
18
HN
HN
79%
89%
H3CO
H3CO
N
N
NH2
d
N
1l
O
64%
Scheme 5. Reagents and conditions: (a) NaOH, H2O, AcOEt, isopropenyl chloroformate, rt, 3 h; (b) NaH, 5, rt, 20 h; (c) PdCl2(dppf)CH2Cl2, NaHCO3, DME/H2O 3.5: 1, reflux,
45 min; (d) TFA, CH2Cl2, reflux, 34 h.
substituents were introduced at position 6 of the azaindole system.
As the introduction of a phenyl moiety resulted in a ten-fold in-
crease of activity (1b, IC50 = 0.27 lM), variously substituted aro-
by the binding of the ligands (induced fit). All the examined struc-
tures were bound in the nicotinamide binding site in a very similar
way, with the azaindole ring inserted between the Y246 and H201
matic rings were linked to carbon 6 (compounds 1c–h). In
residues, interacting with Y246 through a strong p-stacking inter-
general, these compounds showed good inhibitory activity, compa-
action, with a distance between the centers of mass of the two
rings equal to 3.81 Å for 1l. As planned, the carboxamide group
forming the ‘pseudo-cycle’ gives rise to three hydrogen bonds:
one with the S243 hydroxyl (1.91 Å) and two with the G202 back-
bone (1.82 and 2.03 Å with NG202 and OG202, respectively). Con-
sidering the most active product (1l, Fig. 3), the pattern of
interaction with PARP-1 is completed by a hydrogen bond between
the charged nitrogen of the piperidine ring and the hydroxyl group
of Y228, with a distance of 2.04 Å.
The possibility to interact with the Y228 residue is a feature
accessible by those compounds (like e.g., 1l and 1n) with a
charged residue extending within the binding site of the enzyme,
toward the external loops, contributing to increase the affinity of
these ligands. This interaction becomes clearly impossible in
cases where the side chain is too short (e.g., 1p and 1c) or con-
formationally too much constrained (e.g., 1f) to reach the external
loops, or when the compound lacks charged substituents (like 1c).
In these cases the compounds (although correctly inserted) are
not able to interact optimally with the residues of the binding
site, as shown in Figure 4 for compounds 1c, 1f and 1p. In this
latter case the contribution of some water molecules present in
the binding site may explain the relative good affinity with the
enzyme, a contribution which, however, was not taken into ac-
count in this study.
rable to that of 1b, with IC50 values ranging from 0.27 to 1.12
The introduction of an ortho-substituted aromatic ring (compound
1g) led to a decrease in the inhibitory activity (IC50 = 3 M). The
lM.
l
introduction of a further phenyl ring (compound 1f) was also det-
rimental, resulting in complete loss of the enzymatic activity (no
activity up to 5 lM).
Successively, aliphatic aminomethyl groups were added at para
position of the phenyl ring (compounds 1k, l), with the aim of
improving solubility and picking up additional interactions in the
adenosine binding pocket. Both compounds retained a significant
activity against the enzyme, with 1l displaying IC50 = 0.07 lM.
The introduction of an amido group in place of the amino group
(1j vs 1l) led to a decrease of activity.
The insertion of a methylene spacer between the azaindole core
and the carboxamide moiety (1n vs 11) resulted in a loss of enzy-
matic activity, thus confirming the crucial role of the nitrogen-
linked carboxamido group.
The effect of the distance between the basic amine and the core
scaffold was also investigated. Accordingly, compounds 1p–r were
prepared by directly linking the amine nitrogen to the azaindole
nucleus. Whereas compounds 1p–q maintained a good activity,
1r showed a decrease in inhibitory activity (IC50 = 3 lM).
2.3. Molecular modelling studies
2.4. Sensitivity of BRCA1/2 defective tumor cell lines to PARP
inhibitors
The orientations of the described systems inside the binding
site were obtained by using molecular docking experiments that
were further optimised using the QM/MM mixed approach on
the resulting complexes. This QM/MM mixed approach allowed
us to obtain a more complete description of the interactions of
the studied compounds with the human PARP-1 system, as well
as an estimate of the structural changes induced in the complex
PARP inhibitors were tested in vitro for their antiproliferative
activity and compared to the reference compounds Olaparib and
ABT 888 against human tumor cell lines HCC1937 and Capan 1,
characterized by BRCA1 and BRCA2 gene mutation, respectively.
The cell sensitivity assays (Table 2) indicated that compounds

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R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
Table 1
Inhibitory activity of synthesized compounds against recombinant human PARP-1
N
R
N
CONH2
Compd
R
PARP-1 activity (enzymatic assay) IC50 (lM)
Olaparib
MK-4827
ABT-888
1a
0.0045
0.0323
0.0052
2.5
H
1b
1c
1d
1e
1f
1g
Ph
0.27
3.76
0.88
0.27
>5
4-(OCH3)Ph
4-(CH3)2NPh
4-FPh
4-PhPh
2-FPh
3.0
O
F
N
1h
1i
1.12
0.93
N
O
H
N
1j
2.9
N
O
1k
1l
2.10
0.07
1.56
0.28
0.63
O
N
N
N
N
N
1m
1n
1o
N
1p
1q
0.22
0.77
N
N
N
N
1r
3.00
>5
11
1a–f and 1p were inactive in HCC1937 cells, as no inhibition of pro-
liferation was observed up to 200 M drug concentration. In the
HCC1937 cell line, the most promising compounds in terms of
proliferation inhibition appeared to be 1k, 1l, 1n, and 1o, the IC50
values being around 10–20 M. Compounds 1a, 1b and 1d were
inactive also in Capan 1 cells, meanwhile compound 1c, 1e and
l
l

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
1095
Figure 3. Representation of 1l binding mode with the key residues highlighted. Ligand is represented as stick and coloured by atoms. PARP-1 residues are rendered in
wireframe.
Figure 4. Representation of 1l, 1c, 1p and 1f binding modes with the key residues highlighted. Ligands are represented as stick and coloured by atoms. PARP-1 residues are
rendered in wireframe.
1f displayed a poor antiproliferative activity. In both cell lines, the
antiproliferative effect of 1k, 1l, 1n and 1o was in the range of
20 lM. The antiproliferative activity of such compounds was lower
than that of Olaparib but higher than that of ABT-888. Therefore,
given cell sensitivity data and the profile of PARP inhibition we fo-
cused on compound 1l for further studies.
being around 4. Since a marked resistance of the A2780/Dx cells
to Olaparib was observed (resistance index around 40), compound
1l appears to be poorly recognized by Pgp. The activity of the com-
pound on multi-drug resistant cells is a promising feature support-
ing further development of such a PARP inhibitor.
Cell sensitivity to compound 1l was also evaluated on the ovar-
ian carcinoma parental A2780 cells and on the drug-resistant var-
iant A2780/Dx characterized by a multidrug-resistant phenotype
associated to overexpression of P-glycoprotein (Pgp) (Table 3). In
drug resistant cells a slightly reduced sensitivity to compound 1l
was observed as compared to parental cells, the resistance index
2.5. Assessment of the inhibitory activity on a cellular
PARylation assay
Compound 1l showed to be an active inhibitor also through a
functional PARylation assay on a cellular model (HeLa human
endometrial carcinoma cell line). HeLa cells are usually used for

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R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
Table 2
3. Conclusions
Cell sensitivity of cells with BRCA1 (HCC1937) and BRCA2 (Capan 1) gene mutationsa
Compd
HCC1937; IC50
(l
M
SD)
Capan 1; IC50
(
lM
SD)
In conclusion, we have synthesized a series of 6-substituted
pyrrolo[2,3-b]pyridine-1-carboxamides (7-azaindoles) in which
the pyridine- nitrogen atom locks the N-carboxamide group within
a six-membered ‘pseudo-cycle’ through an intramolecular hydro-
gen bond. Molecular modelling studies support that this interac-
tion constrains the group into the optimal binding orientation.
The generation of 6-substituted derivatives with aromatic or het-
erocyclic groups allowed us to identify the 7-azaindole as a new
scaffold for PARP inhibition.
Olaparib
ABT-888
1a
1b
1c
1d
1e
1f
1g
10.3 5.4
97.16 32
>200
>200
>200
>200
>200
>200
n.d.
6.3 2.5
39.7 4.7
>200
>200
161.9 35.1
>200
61.1 19.6
81.8 1.9
n.d.
1h
n.d.
n.d.
The compounds displayed a variable pattern of target inhibition
profile that in part paralleled the antiproliferative activity in cell
lines characterized by homologous recombination defects.
Among the compounds, the promising features of 1l
(ST7710AA1), emerged based on in vitro target inhibition, cell sen-
sitivity and capability to substantially bypass the multidrug resis-
tance mediated by Pgp. In antitumor activity studies against the
MX1 human breast carcinoma growth in nude mice, compound
11 exhibited an effect similar to that of Olaparib in terms of tumor
volume inhibition when used at a lower dose than the reference
compound. Treatment was well tolerated, as no deaths or signifi-
cant weight losses were observed among the treated animals.
All these results suggest that 7-azaindole-1-carboxamides can
be considered a new series of PARP-1 inhibitors with potent
in vitro and in vivo activity, which are worth of further develop-
ment toward a candidate for tumor treatment.
1i
1j
1k
1l
1m
1n
1o
1p
1q
61.43 15.02
72.33 18.9
17.24 4.4
19.43 4.2
41.21 4.9
14.98 1.56
13.45 3.0
>200
87.07 42.9
24.4 12.0
15.90 8.9
22.04 9.2
54.52 22.9
21.69 5.9
19.71 11.9
83.8 7.8
138.4 27.0
n.d.
110.67 43.3
n.d.
182.4 4.2
1r
11
182.4 4.2
a
Cell sensitivity was evaluated by growth inhibition assays based on cell
counting. Cells were seeded and 24 h later they were exposed to the compounds for
72 h. At the end of treatment, cells were counted using a cell counter.
Table 3
Cell sensitivity to compound 1l and Olaparib of cell lines overexpressing Pgpa
Compd
A2780 ovarian
ca.
A2780/Dx (Pgp) ovarian
ca.
Resistance
index
4. Experimental section
IC50, lM
41.9
11.2
4.1. General experimental method
Olaparib 1.00
1l 2.90
41.9
3.9
All reagents and solvents were reagent grade or were purified
by standard methods before use. Melting points were determined
in open capillaries and are uncorrected. NMR spectra were re-
corded at 300 MHz. Chemical shifts (d values) and coupling con-
stants (J values) are given in ppm and Hz, respectively. Solvents
were routinely distilled prior to use; anhydrous tetrahydrofuran
(THF) and ether (Et2O) were obtained by distillation from so-
dium-benzophenone ketyl; dry methylene chloride was obtained
by distillation from phosphorus pentoxide. All reactions requiring
anhydrous conditions were performed under a positive nitrogen
flow, and all glassware were oven dried and/or flame dried. Isola-
tion and purification of the compounds were performed by flash
column chromatography on silica gel 60 (230–400 mesh). Analyt-
ical thin-layer chromatography (TLC) was conducted on Fluka
TLC plates (silica gel 60 F254, aluminum foil).
a
Cell sensitivity was evaluated after 72 h exposure. Results are expressed as IC50
lM), and the relative standard deviations were lower than 10%. Resistance index is
(
the ratio between IC50 of resistant and sensitive cells.
this functional assay because they represent the most suitable
model to measure, in a simple and efficient way, the ability of
tested compounds to affect the PARP-mediated PARylation of nu-
clear proteins, following a strong DNA damage induced by treat-
ment with H2O2.29
As shown in Figure 5, the extent of PARylated proteins de-
creased in a dose-dependent manner in cells pre-treated with com-
pound 1l, although to lesser extent (EC50 = 400 nM) with respect to
Olaparib (EC50 = 2.5 nM), as expected on the basis of their relative
activity against recombinant PARP-1 enzyme (Table 1). Only very
marginal fluorescent signals were measured at the highest dose,
thus confirming the ability of compound 1l to target and inhibit
PARP-1 activity.
Compounds 3,30 421 and 521 were synthesized according to lit-
erature procedures.
4.2. Pyrrolo[2,3-b]pyridine-1-carboxylic acid amide (1a)
2.6. Evaluation of in vivo antitumor activity
To a solution of triphosgene (503 mg, 1.70 mmol) in dichloro-
methane (5 mL) cooled to 0 °C compound 2 (500 mg, 4.23 mmol)
dissolved in dichloromethane (10 mL), DIPEA (1.6 mL, 9.30 mmol)
and NH3/CH2Cl2 (5 mL saturated solution) were dropped sequen-
tially. The mixture was stirred at room temperature for 2 h, then
it was washed twice with 0.25 M HCl, twice with saturated
NaHCO3 solution, then dried and evaporated. The residue was puri-
fied by flash chromatography (CH2Cl2: acetone 99:1) to obtain the
title compound as a white solid. Yield 20%; mp 169 °C; 1H NMR
(CDCl3) d 9.68 (1H, br s); 8.33 (1H, dd, J = 4.9, 1.8 Hz); 8.00 (1H,
d, J = 3.7 Hz); 7.95 (1H, dd, J = 8.2, 1.8 Hz); 7.23 (1H, dd, J = 8.2,
4.9 Hz); 6.59 (1H, d, J = 3.7 Hz); 5.55 (1H, br s).
Antitumor activity of compound 1l, delivered i.p. and adminis-
tered every 2 days for 2 weeks (qd2/wx2w), was examined on the
in vivo breast carcinoma line MX1, a triple-negative human breast
tumor carrying BRCA1 deletion and BRCA2 mutation, that grows in
athymic (SCID beige) mice as subcutaneous (s.c.) tumor.
A significant antitumor effect was observed following adminis-
tration of a dose 100 mg/kg of compound 11 (Table 4). When com-
pared to Olaparib (167 mg/kg) administered with the same
schedule, compound 1l displayed a similar effect in terms of tumor
volume inhibition (TVI, around 35%). Treatment was well tolerated,
as no deaths or significant weight losses were observed among the
treated animals.

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
1097
Figure 5. Effect of 1l and Olaparib on nuclear protein PARylation in HeLa (human endometrial ca.) cells. Cells were pre-treated with serial dilutions of test compounds for
18 h at 37 °C. DNA damage was then provoked by addition of H2O2 500 M. As negative control, cells untreated with H2O2 were used. Protein PARylation was detected in fixed
cells by using the primary PAR mAb (Alexis) and the secondary anti-mouse Alexa Fluor 488 antibody (Molecular probes). Fluorescence signals, related to the residual amount
of PAR polymer on nuclear proteins, were read on the HCS system Operetta. Nuclei were stained with 5 M Draq5 (Alexis). The percent of PAR-positive cells was calculated at
l
l
last by measuring the ratio between the numbers of PAR-positive nuclei over the total number of Draq5-labeled nuclei.
Table 4
Antitumor activity of 1l in comparison with Olaparib against MX1 breast carcinoma
Compd
Dose/route; mg/kg
Schedule
BWL %
Lethality
TV SE (d + 38)
TVI % (d + 38)
Vehicle
1l
Olaparib
0
q2d/wx2w
q2d/wx2w
q2d/wx2w
0
1
0
0/8
0/8
0/8
781 296
502 32
511 64
100/ip
167ip
*36
*35
Treatments started 7 days after tumor injection.
*
P <0.05 versus vehicle-treated group (Mann–Whitney’s test).
Anal. Calcd for C8H7N3O: C, 59.62; H, 4.38; N, 26.07. Found: C,
59.75; H, 4.42; N, 26.17.
J = 8.2 Hz); 7.65 (1H, d, J = 8.1 Hz); 7.50–7.37 (2H, m); 6.60 (1H,
d, J = 3.8 Hz); 5.63 (1H, br s); 3.73–3.63 (2H, m), 2.99–2.81 (8H,
m); 2.70 (3H, br s). Anal. Calcd for C20H23N5O: C, 68.74; H, 6.63;
N, 20.04. Found: C, 69.00; H, 6.65; N, 20.07.
4.3. Suzuki–Miyaura reactions. General procedure A31
To a degassed 3.5:1 mixture of dimethoxyethane and water
(2 mL), compound 5 (0.5 mmol), the appropriate boronic acid
(1 mmol), PdCl2(dppf)CH2Cl2 (0.025 mmol) and sodium bicarbon-
ate (1.5 mmol) were added under nitrogen. The mixture was
heated for 1–4 h. The layers were separated and the organic phase
was evaporated. Purification by flash chromatography gave the de-
sired compounds. Following this procedure compounds 1m–n, 6b–
e, 6g–h, 6j, 6l–m, 11, 15 were prepared.
4.3.2. 6-(4-Pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-
b]pyridine-1-carboxylic acid amide (1n)
From compound 9. Heated 1 h at 110 °C. Flash chromatography
(CH2Cl2:MeOH 92:8). Yield 29%; white solid. mp 168 °C; 1H NMR
(CDCl3) d 9.73 (1H, br s); 8.04–7.94 (4H, m); 7.77 (2H, d,
J = 7.3 Hz); 7.66 (1H, d, J = 8.4 Hz), 6.60 (1H, d, J = 3.4 Hz); 5.63
(1H,br s); 4.17 (2H, s); 3.39–2.62 (4H, m); 2.33–2.12 (4H, m). Anal.
Calcd for C19H20N4O: C, 71.23; H, 6.29; N, 17.49. Found: C, 71.35; H,
6.27; N, 17.42.
4.3.1. 6-[4-(4-Methyl-piperazin-1-ylmethyl)-phenyl]-
pyrrolo[2,3-b]pyridine-1-carboxylic acid amide (1m)
4.3.3. 6-Phenyl-1H-pyrrolo[2,3-b]pyridine (6b)
From compound 9. Heated 3.5 h at 80 °C. Flash chromatography
(CH2Cl2:MeOH 92:8). Yield 46%; white solid; mp 151 °C; 1H NMR
(CDCl3) d 9.80 (1H, br s); 8.05–7.97 (2H, m); 7.93 (2H, d,
Heated 2 h at 110–115 °C. Purification by flash chromatography
(hexane: ethyl acetate 9:1). Yield 58%; white solid; mp 136 °C; 1H
NMR (DMSO-d6) d 11.70 (1H, br s), 8.09 (2H, d, J = 7.4 Hz); 8.03 (1H,

1098
R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
d, J = 8.2 Hz); 7.65 (1H, d, J = 8.2 Hz); 7.53–7.44 (3H, m); 7.38 (1H,
m); 6.47 (1H, m). Anal. Calcd for C13H10N2:C, 80.39; H, 5.19; N,
14.42. Found: C, 80.44; H, 5.21; N, 14.36.
m); 7.47 (1H, d, J = 8.4 Hz); 7.34–7.18 (2H, m); 6.50 (1H, m);
3.97–3.73 (4H, m); 3.88–3.72 (2H, m); 3.54–3.34 (2H, m); 1.75
(1H, m); 1.09–0.94 (2H, m); 0.90–0.64 (2H, m). Anal. Calcd for C22-
H21FN4O2: C, 67.33; H, 5.39; N, 14.28. Found: C, 69.86; H, 5.34; N,
13.88.
4.3.4. 6-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (6c)
Heated 2 h at 110–115 °C. Purification by flash chromatography
(hexane:ethyl acetate 7:3). Yield 85%; white solid; mp 185 °C; 1H
NMR (CDCl3) d 10.22 (1H, br s); 8.01 (1H, d, J = 7.8 Hz); 7.99 (2H,
d, J = 8.7 Hz); 7.51 (1H, d, J = 7.8 Hz); 7.26 (1H, m); 7.06 (2H, d,
J = 8.7 Hz); 6.52 (1H, m); 3.91 (3H, s). Anal. Calcd for C14H12N2O:
C, 74.98; H, 5.39; N, 12.49. Found: C, 75.11; H, 5.32; N, 12.53.
4.3.9. Piperidin-1-yl-[4-(1H-pyrrolo[2,3-b]pyridin-6-yl)-
phenyl]-methanone (6j)
Heated 1 h at 110–115 °C. Purification by flash chromatography
(CH2Cl2:MeOH 97:3). Yield 89%; white solid; mp 183 °C; 1H NMR
(CDCl3) d 10.64 (1H, br s); 8.11–7.97 (3H, m); 7.59–7.50 (3H, m);
7.25 (1H, m); 6.50 (1H, m); 3.86–3.66 (2H, m); 3.53–3.29 (2H,
m); 1.78–1.41 (6H, m). Anal. Calcd for C19H19N3O: C, 74.73; H,
6.27; N, 13.76. Found: C, 74.66; H, 6.22; N, 13.60.
4.3.5. Dimethyl-[4-(1H-pyrrolo[2,3-b]pyridin-6-yl)phenyl]-
amine (6d)
Heated 90 min at 110–115 °C. Purification by flash chromatog-
raphy (hexane:ethyl acetate 7:3). Yield 64%; white solid; mp
198 °C; 1H NMR (DMSO-d6) d 11.61 1H, br s); 8.02 (1H, d,
J = 8.7 Hz); 7.96 (2H, d, J = 8.4 Hz); 7.57 (1H, d, J = 8.7 Hz); 7.41
(1H, m); 6.89 (2H, d, J = 8.4 Hz), 6.44 (1H, m); 2.98 (6H, s). Anal.
Calcd for C15H15N3: C, 75.92; H, 6.37; N, 17.71. Found: C, 75.80;
H, 6.31; N, 17.96.
4.3.10. 6-(4-Morpholin-4-ylmethyl-phenyl)-1H-pyrrolo[2,3-
b]pyridine (6k)
Heated 1 h at 110 °C. Purification by flash chromatography
(CH2Cl2: MeOH 95:5). Yield 81%; white solid; mp 170 °C; 1H
NMR (CDCl3) d 10.75 (1H, br s); 8.02 (1H, d, J = 8.4 Hz); 7.98 (2H,
d, J = 7.7 Hz); 7.53 (1H, d, J = 8.4 Hz); 7.49 (2H, d, J = 7.7 Hz); 7.22
(1H, m); 6.50 (1H, m); 3.83–3.67 (4H, m); 3.61 (2H, s); 2.64–2.39
(4H, m). Anal. Calcd for C18H19N3O: C, 73.69; H, 6.53; N, 14.32.
Found: C, 73.51; H, 6.58; N, 14.12.
4.3.6. 6-(4-Fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (6e)
Heated 90 min at 110–115 °C. Purification by flash chromatog-
raphy (hexane:ethyl acetate 8:2). Yield 82%; white solid; mp
216 °C; 1H NMR (CDCl3) d 10.63 (1H, br s); 8.08–7.94 (3H, m);
7.49 (1H, d, J = 8.2 Hz); 7.27–7.14 (3H, m); 6.52 (1H, m). Anal. Calcd
for C13H9FN2: C, 73.57; H, 4.27; N, 13.20. Found: C, 73.79; H, 4.23;
N, 13.44.
4.3.11. 6-(4-Piperidin-1-ylmethyl-phenyl)-1H-pyrrolo[2,3-
b]pyridine (6l)
Heated 1 h at 110–115 °C. Purification by flash chromatography
(CH2Cl2: MeOH 92:8). Yield 68%; white solid; mp 190 °C; 1H NMR
(DMSO-d6) d 11.66 (1H, br s); 8.07–7.92 (3H, m); 7.62 (1H, d,
J = 8.7 Hz); 7.46 (1H, m); 7.37 (2H, d, J = 8.2 Hz); 6.44 (1H, m);
3.45 (2H, s); 2.42–2.27 (4H, m), 1.55–1.31 (6H, m).
Anal. Calcd for C19H21N3: C, 78.32; H, 7.26; N, 14.42. Found: C,
78.49; H, 7.22; N, 14.66.
4.3.7. 6-(2-Fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (6g)
Heated 1 h at 110–115 °C. Purification by flash chromatography
(hexane:ethyl acetate 7:3). Yield 78%; white solid; mp 155 °C; 1H
NMR (CDCl3) d 11.24 (1H, br s); 8.03 (1H, d, J = 8.2 Hz); 7.93 (1H,
t, J = 7.5 Hz); 7.54 (1H, dd, J = 1.9; 8.1 Hz); 7.42 (1H, m); 7.37–
7.15 (3H, m); 6.50 (1H, m). Anal. Calcd for C13H9FN2: C, 73.57; H,
4.27; N, 13.20. Found: C, 73.79; H, 4.22; N, 13.48.
4.3.12. 2-[6-(4-Pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-
b]pyridin-1-yl]-acetamide (11)
From compound 10. Heated 1 h at 110 °C. Crystallized from
CH2Cl2. Yield 29%; white solid; mp 181 °C; 1H NMR (DMSO-d6) d
8.11–7.93 (3H, s); 7.71–7.58 (2H, m); 7.51 (1H, d, J = 3.0 Hz);
7.40 (2H, d, J = 7.6 Hz); 7.23 (1H, br s); 6.48 (1H, d, J = 3.0 Hz);
4.95 (2H, s); 3.61 (2H, s); 2.53–2.39 (4H, m); 1.75–1.65 (4H, m).
Anal. Calcd for C20H22N4O: C, 71.83; H, 6.63; N, 16.75. Found: C,
71.68; H, 6.67; N, 16.61.
4.3.8. (4-Cyclopropanecarbonylpiperazin-1-yl)-[2-fluoro-5-(1H-
pyrrolo[2,3-b]pyridin-6-yl)-phenyl]-methanone (6h)
To a solution of 5 (100 mg, 0.51 mmol) in degassed DME/H2O
3.5:1 (2 mL) under N2 were added 3-carboxy-4-fluorophenylbo-
ronic acid (188 mg, 1.02 mmol), NaHCO3 (128 mg, 1.53 mmol)
and PdCl2(dppf)CH2Cl2 (19 mg, 0.025 mmol). The solution was
heated for 2 h at 80 °C and for 2 h at 110 °C in the dark. After addi-
tion of ethyl acetate, the mixture was washed with 1 N HCl. The
aqueous phase was extracted three times with ethyl acetate. The
collected organic layers were dried, filtered and evaporated to give
a crude (252 mg) that was suspended in CH2Cl2 (10 mL) and meth-
anol (0.5 mL). The resulting mixture was refluxed for 30 min.,
cooled and filtered to give 2-fluoro-5-(1H-pyrrolo[2,3-b]pyridin-
6-yl)-benzoic acid as a pale yellow sticky solid (70 mg, 54%); 1H
NMR (DMSO-d6) d 11.78 (1H, br s); 8.67 (1H, dd, J = 2.0, 7.5 Hz);
8.33 (1H, m); 8.06 (1H, d, J = 8.2 Hz); 7.70 (1H, d, J = 8.2 Hz); 7.53
(1H, m); 7.41 (1H, m); 6.49 (1H, m).
4.3.13. 6-(4-Dimethylaminomethylphenyl)-pyrrolo[2,3-
b]pyridine-1-carboxylic acid bis-(4-methoxybenzyl)amide (15)
From compound 14. Heated for 3 h at 100 °C. Flash column
chromatography (CH2Cl2:MeOH 95:5). Yield 57%; sticky solid; 1H
NMR (CDCl3) d 8.05–7.92 (3H, m); 7.67 (1H, d, J = 8.1 Hz); 7.61
(1H, d, J = 3.7 Hz); 7.56–7.47 (2H, m); 7.41 (1H, m); 7.30–7.10
(3H, m); 6.92–6.79 (4H, m); 6.62 (1H, d, J = 3.7 Hz); 4.61 (4H, s);
3.94 (2H, s); 3.81 (6H, s); 2.61 (6H, s). Anal. Calcd for
C33H34N4O3: C, 74.13; H, 6.41; N, 10.48. Found: C, 74.28; H, 6.45;
N, 10.59.
To a solution of the above compound (68 mg, 0.27 mmol) in
anhydrous DMF (2 mL) under N2 at 0 °C were added WSC (61 mg,
0.32 mmol) and HOBt (43 mg, 0.32 mmol). The resulting mixture
32
4.4. 6-Biphenyl-4-yl-1H-pyrrolo[2,3-b]pyridine (6f)
was stirred for 30 min, then it was added with DIPEA (141
0.81 mmol) and 1-(cyclopropylcarbonyl)piperazine (115
l
l
l,
l,
A flask charged with 4-bromobiphenyl (100 mg, 0.43 mmol),
bispinacolate diboron (100 mg, 0.47 mmol), PdCl2(dppf)CH2Cl2
(9.4 mg, 0.01 mmol), KOAc (126 mg, 1.29 mmol) in dioxane
(2.60 mL) was heated for 5 h at 100 °C under nitrogen. After cooling
0.81 mmol). Stirring was continued at room temperature for
20 h. The residue was partitioned between water and ethyl acetate
and the phases were separated. The organic layer was washed with
brine, dried, filtered and evaporated. Purification by flash chroma-
tography (CH2Cl2:MeOH 95:5) afforded 85 mg (80%) of the title
compound. 1H NMR (CDCl3) d 10.88 (1H, br s); 8.18–7.95 (3H,
the solution to room temperature, compound
5 (169 mg,
0.86 mmol), PdCl2(dppf)CH2Cl2 (9.4 mg, 0.013 mmol), 2 M Na2CO3
(0.54 mL, 1.07 mmol) were added and the mixture was stirred at
100 °C for 4 h. The solution was cooled to room temperature and

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
1099
the product was extracted with ethyl acetate. The organic layer was
4.7. General procedure C
washed with 0.25 M HCl, dried, filtered and evaporated. Purification
by flash chromatography (hexane:ethyl acetate 85:15) gave 6f (57%)
as a white solid; mp 245 °C; 1H NMR (DMSO-d6) d 11.72 (1H, br s);
8.19 (2H, d, J = 7.9 Hz); 8.02 (1H, d, J = 8.4 Hz); 7.84–7.60 (5H, m);
7.55–7.32 (4H, m); 6.46 (1H, m). Anal. Calcd for C19H14N2: C,
84.42; H, 5.22; N, 10.36. Found: C, 84.66; H, 5.18; N, 10.48.
Compound 6 (2.5 mmol) was added to a solution of triphosgene
(1 mmol) and DIPEA (5.5 mmol) in toluene (0.35 mL). The mixture
was heated at 80 °C for 1.5–3 h, then it was cooled to room temper-
ature and NH3/CH2Cl2 (3 mL saturated solution) was added. After
stirring overnight at room temperature, ethyl acetate was added
and the organic phase was washed twice with 0.25 M HCl, twice
with saturated NaHCO3 solution, then dried and evaporated. The
residue was purified by flash chromatography.
4.5. 2-(1H-Pyrrolo[2,3-b]pyridin-6-yl)-pyrrole-1-carboxylic acid
33
tert-butyl ester (6i)
Following this procedure compounds 1b–f, 1i, 1p were
prepared.
A nitrogen purged mixture of 5 (200 mg, 1.01 mmol), N-Boc-2-
pyrroleboronic acid (321 mg, 1.52 mmol), K2CO3 (279 mg,
2.02 mmol) and Pd(Ph3)4 (58.4 mg, 0.05 mmol) in DME:/H2O 4:1
(5.5 mL) was stirred at 100 °C for 3 h in the dark. The mixture
was diluted with CH2Cl2 and washed with aqueous NH4Cl. The
aqueous phase was re-extracted with CH2Cl2 and the combined or-
ganic extracts were dried over Na2SO4 and concentrated in vacuo.
Purification by flash chromatography (hexane:diethyl ether 6:4)
afforded 6i (84%) as a light yellow solid; mp 136 °C; 1H NMR
(CDCl3) d 12.00 (1H, br s); 7.95 (1H, d, J = 8.1 Hz); 7.45 (1H, m);
7.27–7.16 (2H, m); 6.51–6.43 (2H, m); 6.33 (1H, m); 1.18 (9H, s).
Anal. Calcd for C16H17N3O2: C, 67.83; H, 6.05; N, 14.83. Found: C,
67.99; H, 6.00; N, 14.97.
4.7.1. 6-Phenyl-pyrrolo[2,3-b]pyridine-1-carboxylic acid amide
(1b)
Heated 90 min at 80 °C. Flash chromatography (hexane:ethyl
acetate 75:25). Yield 46%; white solid; mp 205 °C. 1H NMR (CDCl3)
d 9.86 (1H, br s); 8.01–7.91 (4H, m); 7.69 (1H, d, J = 8.2 Hz); 7.58–
7.41 (3H, m); 6.61 (1H, d, J = 3.8 Hz); 5.63 (1H, br s). Anal. Calcd for
C14H11N3O: C, 70.87; H, 4.67; N, 17.71. Found: C, 70.64; H, 4.63; N,
17.91.
4.7.2. 6-(4-Methoxy-phenyl)-pyrrolo[2,3-b]pyridine-1-
carboxylic acid amide (1c)
Heated 2 h at 80 °C. Flash chromatography (hexane: acetone
75:25). Yield 29%; white solid; mp 199 °C; 1H NMR (CDCl3) d
9.88 (1H, br s); 8.02–7.88 (4H, m); 7.62 (1H, d, J = 8.4 Hz); 7.04
(2H, d, J = 8.6 Hz); 6.59 (1H, d, J = 4.4 Hz); 5.58 (1H, br s); 3.90
(3H, s). Anal. Calcd for C15H13N3O2: C, 67.40; H, 4.90; N, 15.72.
Found: C, 67.61; H, 4.95; N, 15.66.
4.6. General procedure B25
To a solution of 7-hydroxy-lH-pyrrolo[2,3-b]pyridinium 3-chloro-
benzoate (5.15 mmol.)30 and dimethyl sulfate (5.67 mmol) in anhy-
drous MeCN (10 mL) were stirred overnight under N2 at 55–60 °C.
Aftercooling to room temperature, theamine (25.5 mmol) wasadded
and the mixture was stirred overnight under N2 at 55–60 °C. After
cooling toroom temperature, the suspension wasconcentratedunder
reduced pressure and the residue partitioned between CH2Cl2
(10 mL) and 10% aqueous Na2CO3 (2.5 mL). The aqueous phase was
extracted with CH2Cl2 (3 Â 5 mL) and the combined organic layers
were washed with 10% aqueous Na2CO3 (3 mL), brine (3 mL), H2O
(3 mL) and dried over Na2SO4 and evaporated under reduced pres-
sure. The crude was purified by flash column chromatography. Fol-
lowing this procedure compounds 6p–r were prepared.
4.7.3. 6-(4-Dimethylamino-phenyl)-pyrrolo[2,3-b]pyridine-1-
carboxylic acid amide (1d)
Stirred 2 h at room temperature. Flash chromatography (dichlo-
romethane:acetone 99:1). Yield 46%; white solid; mp 222 °C; 1H
NMR (DMSO-d6) d 9.26 (1H, br s); 8.10 (1H, br s); 8.08 (1H, d,
J = 8.2 Hz); 7.90 (2H, d, J = 9.1 Hz); 7.85 (1H, d, J = 4.1 Hz); 7.75
(1H, d, J = 8.2 Hz); 6.82 (2H, d, J = 9.1 Hz); 6.65 (1H, d, J = 4.1 Hz);
2.76 (6H, s). Anal. Calcd for C16H16N4O: C, 68.55; H, 5.75; N,
19.99. Found: C, 68.70; H, 5.98; N, 20.08.
4.6.1. 6-Pyrrolidin-1-yl-1H-pyrrolo[2,3-b]pyridine (6p)
4.7.4. 6-(4-Fluoro-phenyl)-pyrrolo[2,3-b]pyridine-1-carboxylic
acid amide (1e)
Heated for 12 h at 55–60 °C, then 8 h at 50–55 °C. Flash column
chromatography (hexane:ethyl acetate 7:3). Yield 56%; white so-
lid; mp 218 °C; 1H NMR (DMSO-d6) d 10.98 (1H, br s); 7.63 (1H,
d, J = 8.4 Hz); 6.91 (1H, m); 6.23 (1H, d, J = 8.4 Hz), 6.17 (1H, m);
3.35–3.28 (4H, m); 1.99–1.84 (4H, m). Anal. Calcd for C11H13N3:
C, 70.56; H, 7.00; N, 22.44. Found: C, 70.41; H, 7.04; N, 22.22.
Heated 2.5 h at 80 °C. Flash chromatography (hexane:ethyl ace-
tate 80:20). Yield 85%; white solid; mp 174 °C; 1H NMR (CDCl3) d
9.75 (1H, br s), 8.01 (1H, d, J = 8.1 Hz); 7.99–7.88 (3H, m); 7.61
(1H, d, J = 8.1 Hz); 7.19 (2H, t, J = 8.8 Hz); 6.59 (1H, d, J = 4.4 Hz);
5.62 (1H, br s). Anal. Calcd for C14H10FN3O: C, 65.88; H, 3.95; N,
16.46. Found: C, 65.65; H, 3.99; N, 16.67.
4.6.2. 6-(4-Methyl-piperazin-1-yl)-1H-pyrrolo[2,3-b]pyridine
(6q)
4.7.5. 6-Biphenyl-4-yl-pyrrolo[2,3-b]pyridine-1-carboxylic acid
amide (1f)
Heated for 12 h at 55–60 °C, then 12 h at 50–55 °C. Flash col-
umn chromatography (CH2Cl2:MeOH 92:8). Yield 29%; white solid;
mp 197 °C; 1H NMR (DMSO-d6) d 11.00 (1H, br s); 7.67 (1H, d,
J = 8.6 Hz); 7.02 (1H, m); 6.60 (1H, d, J = 8.6 Hz); 6.19 (1H, m);
3.46–3.37 (4H, m); 2.42–2.35 (4H, m); 2.20 (3H, s). Anal. Calcd
for C12H16N4: C, 66.64; H, 7.46; N, 25.90. Found: C, 66.51; H,
7.42; N, 26.01.
Heated 3 h at 80 °C. Flash chromatography (hexane:ethyl ace-
tate 80:20), then crystallization from diethyl ether. Yield 76%;
white solid; mp 235 °C; 1H NMR (DMSO-d6) d 9.16 (1H, br s);
8.26–8.12 (4H, m), 7.98–7.91 (2H, m), 7.83 (2H, d, J = 8.2 Hz);
7.75 (2H, d, J = 7.4 Hz); 7.54–7.45 (2H, m); 7.39 (1H, t, J = 7.0 Hz);
6.73 (1H, d, J = 4.0 Hz). Anal. Calcd for C20H15N3O: C, 76.66; H,
4.82; N, 13.41. Found: 76.49; H, 4.86; N, 13.61.
4.6.3. 6-Piperidin-1-yl-1H-pyrrolo[2,3-b]pyridine (6r)
Heated for 12 h at 55–60 °C, then 8 h at 50–55 °C. Flash column
chromatography (Hexane:ethyl acetate 8:2). Yield 35%; sticky so-
lid; 1H NMR (CDCl3) d 8.72 (1H, br s); 7.73 (1H, d, J = 8.5 Hz);
6.98 (1H, m); 6.60 (1H, d, J = 8.5 Hz); 6.33 (1H, m); 3.60–3.48
(4H, m); 1.75–1.60 (6H, m). Anal. Calcd for C12H15N3: C, 71.61; H,
7.51; N, 20.88. Found: C, 71.87; H, 7.55; N, 20.66.
4.7.6. 6-(1H-Pyrrol-2-yl)-pyrrolo[2,3-b]pyridine-1-carboxylic
acid amide (1i)
Heated 1 h at 80 °C. Flash chromatography (dichlorometh-
ane:acetone 197:3) gave 2-(1-carbamoyl-1H-pyrrolo[2,3-b]pyri-
din-6-yl)-pyrrole-1-carboxylic acid tert-butyl ester. Yield 74%;
white solid; mp 186 °C. 1H NMR (CDCl3) d 9.58 (1H, br s);

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R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
7.99–7.90 (2H, m); 7.38 (1H, m); 7.34 (1H, d, J = 8.1 Hz); 6.56 (1H,
m); 6.43 (1H, m); 6.27 (1H, m); 5.42 (1H, br s); 1.32 (9H, s).
Removal of Boc protecting group was accomplished by refluxing
a solution of the above compound (40 mg, 0.123 mmol) in MeOH
(1.5 mL)/10.2 N HCl (0.016 mL) for 5 h. Methanol was evaporated
and the residual slurry was added with CH2Cl2. The organic phase
was washed with 10% Na2CO3, dried, filtered and evaporated. Puri-
fication by flash chromatography (CH2Cl2: acetone 95:5) gave 1i
(59%); 1H NMR (DMSO-d6) d 11.75 (1H, br s); 8.92 (1H, br s);
7.98 (1H, d, J = 8.2 Hz); 7.91 (1H, br s); 7.81 (1H, d, J = 3.8 Hz);
7.60 (1H, d, J = 8.2 Hz); 6.92 (1H, m); 6.79 (1H, d, J = 3.9 Hz); 6.59
(1H, m); 6.15 (1H, d, J = 4.0 Hz). Anal. Calcd for C12H10N4O: C,
63.71; H, 4.46; N, 24.77. Found: C, 63.88; H, 4.50; N, 24.61.
4.8.6. 6-Piperidin-1-yl-pyrrolo[2,3-b]pyridine-1-carboxylic acid
4-nitro-phenyl ester (7r)
Heated for 3.5 h at 75 °C. Used for the next step without further
purification.
4.8.7. 6-Bromo-pyrrolo[2,3-b]pyridine-1-carboxylic acid 4-
nitro-phenyl ester (8)
.From compound 5. Stirred for 1 h at room temperature. Used
without further purification.
4.9. General procedure E
To a solution of 7 (1 mmol) in dry DMF (3.6 mL) (NH4)2CO3
(1.6 mmol) was added and the mixture was heated at 40–60 °C
for 0.5–2.5 h. After addition of water (10 mL) the mixture was ex-
tracted with EtOAc (3 Â 10 mL). The combined organic layers were
washed with brine (50 mL), dried over Na2SO4, filtered and evapo-
rated. Following this procedure compounds 1g–h, j, k, q, r, 9 were
prepared.
4.7.7. 6-Pyrrolidin-1-yl-pyrrolo[2,3-b]pyridine-1-carboxylic
acid amide (1p)
Heated for 1 h at 80 °C in toluene. Flash column chromatogra-
phy (hexane:ethyl acetate 75:25). Yield 80%; white solid; mp
198 °C; 1H NMR (DMSO-d6) d 9.11 (1H, br s); 7.83 (1H, m); 7.77
(1H, d, J = 8.9 Hz); 7.45 (1H, d, J = 4.0 Hz); 6.43 (1H, br s); 6.41
(1H, d, J = 4.0 Hz); 3.42–3.32 (4H, m); 2.01–1.90 (4H, m). Anal.
Calcd for C12H14N4O: C, 62.59; H, 6.13; N, 24.33. Found: C, 62.39;
H, 6.18; N, 24.45.
4.9.1. 6-(2-Fluoro-phenyl)-pyrrolo[2,3-b]pyridine-1-carboxylic
acid amide (1g)
Heated 1 h at 60 °C. Flash column chromatography (hex-
ane:ethyl acetate 4:3) then crystallization from diethyl ether. Yield
58%; yellow solid; mp 186 °C; 1H NMR (CDCl3) d 9.73 (1H, br s);
8.05–7.98 (2H, m); 7.84 (1H, t, J = 7.6 Hz); 7.69 (1H, d, J = 8.2 Hz);
7.42–7.16 (3H, m); 6.61 (1H, d, J = 4.0 Hz); 5.52 (1H, br s). Anal.
Calcd for C14H10FN3O: C, 65.88; H, 3.95; N, 16.46. Found: C,
65.67; H, 3.92; N, 16.66.
4.8. General procedure D
A
solution of 6 (1 mmol) and 4-nitrophenylchloroformate
(1.6 mmol) in toluene (18 mL, for 7g, h, k, q, r, 8) or CH2Cl2
(18 ml for 7j) containing NaOH (3 mmol) and a catalytic amount
of Bu4NBr (0.05 mmol) was heated (40–100 °C) for 2–7 h under a
stream of nitrogen. Ethyl acetate was added and the organic phase
was washed with water, dried over Na2SO4 and filtered. Following
this procedure compounds 7g, h, j, k, q, r, 8 were prepared.
4.9.2. 6-[3-(4-Cyclopropanecarbonyl-piperazine-1-carbonyl)-4-
fluoro-phenyl]-pyrrolo[2,3-b]pyridine-1-carboxylic acid amide
(1h)
Heated 2.5 h at 60 °C. Flash column chromatography (hexane:
acetone 6:4) then crystallization from CH2Cl2/Et2O. Yield 23%;
white solid; mp 184 °C; 1H NMR (DMSO-d6) d 9.60 (1H, br s);
8.08–7.93 (4H, m); 7.62 (1H, d, J = 8.1 Hz); 7.26 (1H, m); 6.60
(1H, d, J = 4.0 Hz); 5.74 (1H, br s); 3.99–3.57 (6H, m); 3.53–3.30
(2H, m), 1.92 (1H, m); 1.06–0.98 (2H, m), 0.91–0.67 (2H, m). Anal.
Calcd for C23H22FN5O3: C, 63.44; H, 5.09; N, 16.08. Found: C, 63.21;
H, 5.13; N, 15.95.
4.8.1. 6-(2-Fluoro-phenyl)-pyrrolo[2,3-b]pyridine-1-carboxylic
acid 4-nitro-phenyl ester (7g)
Heated for 2 h at 60 °C. Used for the next step without further
purification.
4.8.2. 6-[3-(4-Cyclopropanecarbonyl-piperazine-1-carbonyl)-4-
fluoro-phenyl]-pyrrolo[2,3-b]pyridine-1-carboxylic acid 4-
nitro-phenyl ester (7h)
Heated for 3 h at 100 °C. Used for the next step without further
purification.
4.9.3. 6-[4-(Piperidine-1-carbonyl)-phenyl]-pyrrolo[2,3-
b]pyridine-1-carboxylic acid amide (1j)
Heated 0.5 h at 40 °C. Flash column chromatography (CH2Cl2:
MeOH 197:3). Yield 72%; white solid; mp 179 °C; 1H NMR (CDCl3)
d 9.77 (1H, br s); 8.09–7.93 (4H, m); 7.68 (1H, d, J = 8.1 Hz); 7.52
(2H, d, J = 7.3 Hz); 6.60 (1H, d, J = 3.2 Hz); 5.64 (1H, br s); 3.78–
3.31 (4H, m); 1.94–1.48 (6H, m). Anal. Calcd for C20H20N4O2: C,
68.95; H, 5.79; N, 16.08. Found: C, 69.08; H, 5.72; N, 16.23.
4.8.3. 6-(4-Morpholin-4-ylmethyl-phenyl)-pyrrolo[2,3-
b]pyridine-1-carboxylic acid 4-nitro-phenyl ester (7k)
Heated for 7 h at 40 °C. Used for the next step without further
purification.
4.9.4. 6-(4-Morpholin-4-ylmethyl-phenyl)-pyrrolo[2,3-
b]pyridine-1-carboxylic acid amide (1k)
4.8.4. 6-[4-(Piperidine-1-carbonyl)-phenyl]-pyrrolo[2,3-
b]pyridine-1-carboxylic acid 4-nitro-phenyl ester (7j)
Stirred for 4 h at room temperature. Used for the next step
without further purification.
Heated 3 h at 45 °C. Flash column chromatography (CH2Cl2:
MeOH 96:4). Yield 50%; mp 162 °C; 1H NMR (CDCl3) d 9.82 (1H,
br s); 8.05–7.89 (4H, m); 7.66 (1H, d, J = 7.9 Hz); 7.60–7.35 (2H,
m); 6.59 (1H, d, J = 4.0 Hz); 5.60 (1H, br s); 3.90–3.45 (6H, m);
2.69–2.39 (4H, m). Anal. Calcd for C19H20N4O2: C, 67.84; H, 5.99;
N, 16.66. Found: C, 67.64; H, 5.93; N, 16.83.
4.8.5. 6-(4-Methyl-piperazin-1-yl)-pyrrolo[2,3-b]pyridine-1-
carboxylic acid 4-nitro-phenyl ester (7q)
Heated for 2 h at 100 °C in toluene. Flash column chromatog-
raphy (CH2Cl2:MeOH 95:5). Yield 51%; sticky solid; 1H NMR
(CDCl3) d 8.36 (2H, d, J = 9.1 Hz); 7.74 (1H, d, J = 8.7 Hz); 7.56–
7.48 (3H, m); 6.69 (1H, d, J = 8.7 Hz); 6.54 (1H, d, J = 3.7 Hz);
3.92–3.64 (4H, m); 2.87–2.65 (4H, m); 2.45 (3H, s). Anal. Calcd
for C19H19N5O4: C, 59.84; H, 5.02; N, 18.36. Found: C, 59.66; H,
5.05; N, 18.18.
4.9.5. 6-(4-Methyl-piperazin-1-yl)-pyrrolo[2,3-b]pyridine-1-
carboxylic acid amide (1q)
Heated for 1 h at 45 °C. Flash column chromatography (CH2Cl2:-
MeOH 9:1). Yield 78%; white solid; mp 142 °C; 1H NMR (CDCl3) d
9.34 (1H, br s); 7.75 (1H, d, J = 8.8 Hz); 7.68 (1H, d; J = 3.8 Hz);
6.66 (1H, d, J = 8.8 Hz); 6.40 (1H, d, J = 3.8 Hz); 5.53 (1H, br s);

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
1101
3.75–3.42 (4H, m); 2.91–2.60 (4H, m); 2.45 (3H, s). Anal. Calcd for
13H17N5O: C, 60.21; H, 6.61; N, 27.01. Found: C, 60.08; H, 6.67; N,
26.88.
compound as a white solid; mp 223 °C; 1H NMR (DMSO-d6) d
7.94 (1H, d, J = 8.8 Hz); 7.64 (1H, br s); 7.51 (1H, d, J = 3.5 Hz);
7.26 (1H, d, J = 8.8 Hz); 7.25 (1H, br s); 6.51 (1H, d, J = 3.5 Hz);
4.85 (2H, s). Anal. Calcd for C9H8BrN3O: C, 42.54; H, 3.17; N,
16.54. Found: C, 42.69; H, 3.12; N, 16.36.
C
4.9.6. 6-Piperidin-1-yl-pyrrolo[2,3-b]pyridine-1-carboxylic acid
amide (1r)
Heated for 0.5 h at 40 °C. Flash column chromatography (CH2-
Cl2:MeOH 98:12). Yield 62%; white solid; mp 148 °C; 1H NMR
(CDCl3) d 9.55 (1H, br s); 7.71 (1H, d, J = 8.7 hz), 7.64 (1H, d,
J = 3.8 Hz); 6.66 (1H, d, J = 8.7 Hz); 6.39 (1H, d, J = 3.8 Hz); 5.58
(1H, br s); 3.54–3.50 (4H, m); 1.80–1.60 (6H, m). Anal. Calcd for
4.12. 6-Bromo-pyrrolo[2,3-b]pyridine-1-carboxylic acid bis-(4-
methoxybenzyl)amide (14)
A solution of 8 (138 mg, 0.38 mmol) and bis-(4-methoxyben-
zyl)amine (13)24 (158 mg, 0.61 mmol) in anhydrous DMF
(1.5 mL) was heated at 60 °C for 6 h. After addition of ethyl acetate,
the solution was washed with water and brine, then it was dried
over Na2SO4, filtered and evaporated. Purification by flash column
chromatography (hexane: ethyl acetate 8:2) afforded 109 mg (91%)
of the title compound as a sticky solid; 1H NMR (CDCl3) d 7.78 (1H,
d, J = 8.2 Hz); 7.50 (1H, d, J = 3.8 Hz); 7.35 (1H, d, J = 8.2 Hz); 7.32–
7.15 (4H, m); 6.95–6.80 (4H, m); 6.58 (1H, d, J = 3.8 Hz); 4.55 (4H,
s); 3.80 (6H, s). Anal. Calcd for C24H22BrN3O3: C, 60.01; H, 4.62; N.
8.75.Found: C, 60.21; H, 4.66; N. 8.55.
C13H16N4O: C, 63.91; H, 6.60; N, 22.93. Found: C, 64.08; H, 6.55;
N, 22.73.
4.9.7. 6-Bromo-pyrrolo[2,3-b]pyridine-1-carboxylic acid amide
(9)
From compound 8. Heated 1 h at 50 °C. Quantitative yield;
sticky solid; 1H NMR (CDCl3) d 9.02 (1H, br s); 7.95 (1H, d,
J = 3.9 Hz); 7.81 (1H, d, J = 8.1 Hz); 7.37 (1H, d, J = 8.1 Hz); 6.59
(1H, d, J = 3.9 Hz); 5.59 (1H, br s). Anal. Calcd for C8H6BrN3O: C,
40.03; H, 2.52; N, 17.50. Found: C, 40.23; H, 2.55; N, 17.68.
4.13. 6-(4-Dimethylaminomethyl-phenyl)-pyrrolo[2,3-
4.10. 6-(4-Piperidin-1-ylmethyl-phenyl)-pyrrolo[2,3-
b]pyridine-1-carboxylic acid amide (1l)
b]pyridine-1-carboxylic acid amide (1o)
34
Compound 15 (27 mg, 0.05 mmol) was refluxed in TFA (2 mL)
for 9.5 h. TFA was then removed under vacuum and the residue
was added with CH2Cl2, washed with a saturated solution of
NaHCO3, dried, filtered and evaporated. Purification by PLC (CH2-
Cl2: MeOH 17:3) afforded 11 mg (78%) of the title compound as a
white solid; mp 199 °C; 1H NMR (CDCl3) d 9.81 (1H, br s); 8.04–
7.88 (4H, m); 7.66 (1H, d, J = 8.1 Hz); 7.50 (2H, d, J = 7.9 Hz); 6.59
(1H, d, J = 3.7 Hz); 5.72 (1H, br s); 3.65 (2H, s); 2.40 (6H, s). Anal.
Calcd for C17H18N4O: C, 69.37; H, 6.16; N, 19.03. Found: C, 69.08;
H, 6.21; N, 18.83.
(a) A solution of lithium hexamethyldisilazane in THF (0.28 mL,
1.0 M, 0.28 mmol) was added to
a solution of 6l (75 mg,
0.26 mmol) in THF (3 mL) at À78 °C. The reaction was stirred at
À78 °C for 40 min, whereupon 4-nitrophenylchloroformate
(104 mg, 0.51 mmol) was added portionwise over 5 min., then
the cooling bath was removed and stirring continued for 2 h. Satu-
rated aq. NH4Cl (2 mL) was added and the mixture was extracted
with CH2Cl2 (3 Â 20 mL). The combined organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The residue
was purified by flash chromatography (CH2Cl2: MeOH 95:5) to ob-
tain 1l (17%) as a white solid; mp 177–178 °C; 1H NMR (CDCl3) d
9.82 (1H, br s); 8.03–7.94 (2H, m); 7.90 (2H, d, J = 7.8 Hz); 7.64
(1H, d, J = 8.2 Hz); 7.47 (2H, d, J = 7.8 Hz); 6.58 (1H, d, J = 4.0 Hz);
5.59 (1H, br s); 3.59 (2H, s); 2.58–2.38 (4H, m); 1.75–1.54 (4H,
m); 1.50–1.39 (2H, m). Anal. Calcd for C20H22N4O: C, 71.83; H,
6.63; N, 16.75. Found: C, 71.99; H, 6.603; N, 16.55.
(b) A solution of 19 (1.900 g, 4.2 mmol) in 145 mL of TFA was
refluxed for 34 h. TFA was evaporated under vacuum and the res-
idue was dissolved in ethyl acetate. The organic phase was washed
three times with a cold saturated solution of NaHCO3, then it was
dried, filtered and evaporated. The crude (2.07 g) was first crystal-
lized from AcOEt/Et2O and then purified by flash chromatography
(CH2Cl2: MeOH 95: 5) to obtain the title compound as a white solid
(64%); mp 178 °C. 1H NMR (CDCl3) d 9.82 (1H, br s); 8.03–7.94 (2H,
m); 7.90 (2H, d, J = 7.8 Hz); 7.64 (1H, d, J = 8.2 Hz); 7.47 (2H, d,
J = 7.8 Hz); 6.58 (1H, d, J = 4.0 Hz); 5.59 (1H, br s); 3.59 (2H, s);
2.58–2.38 (4H, m); 1.75–1.54 (4H, m); 1.50–1.39 (2H, m). Anal.
Calcd for C20H22N4O: C, 71.83; H, 6.63; N, 16.75. Found: C, 71.98;
H, 6.72; N, 16.84.
4.14. (4-Methoxybenzyl)carbamic acid isopropenyl ester (17)
To a solution of NaOH (348 mg, 8.7 mmol) in water (4 mL) p-
methoxybenzylamine (0.76 mL, 5.8 mmol) in ethyl acetate
(9.7 mL) was added dropwise, while keeping the temperature be-
low 5 °C. After cooling to 0 °C, isopropenyl chloroformate (1.00 g,
8.2 mmol) was added and the reaction mixture was stirred at
room temperature for 3 h. The layers were separated. The organic
layer was dried over Na2SO4, filtered and evaporated. The crude
product (1.4 g) was used for the next step without further
purification.
4.15. 6-Bromo-pyrrolo[2,3-b]pyridine-1-carboxylic acid 4-
methoxybenzylamide (18)
To a solution of 6-bromo-7-azaindole (0.500 g, 2.5 mmol) in
anhydrous THF (12 mL) under nitrogen, NaH (60% in mineral oil,
0,130 g, 3.25 mmol) was added and the resulting mixture was stir-
red at room temperature for 2 h. A solution of 17 (0.978 g,
4.42 mmol) in anhydrous THF (8 mL) was then added and the mix-
ture was stirred at room temperature for 20 h. The solvent was
evaporated and water was added. The aqueous phase was ex-
tracted with ethyl acetate (3 Â 30 mL). The collected organic layers
were dried, filtered and evaporated to give 1.19 g of a crude that
was purified by column chromatography (Hexane: ethyl acetate
9:1). The title compound was obtained as a white solid (79%);
mp 97 °C; 1H NMR (CDCl3) d 9.47 (1H, br s); 7.98 (1H, d,
J = 3.5 Hz); 7.79 (1H, d, J = 7.8 Hz); 7.45–7.31 (3H, m); 6.91 (2H,
d, J = 8.4 Hz); 6.52 (1H, d, J = 3.5 Hz); 4.60 (2H, m); 3.80 (3H, s).
Anal. Calcd for C16H14BrN3O2: C, 53.35; H, 3.92; N, 11.67. Found:C,
53.56; H, 3.96; N, 11.48.
4.11. 2-(6-Bromo-pyrrolo[2,3-b]pyridin-1-yl)-acetamide (10)
Compound
5 (200 mg, 1.01 mmol) dissolved in dry DMF
(0.61 mL) was added dropwise to a suspension of NaH (60% in min-
eral oil) in dry DMF (0.81 mL) and the mixture was stirred for
30 min at room temperature. Iodoacetamide (187 mg, 1.01 mmol)
dissolved in DMF (1.01 mL) was added dropwise and the reaction
was stirred for 3 h at room temperature. Iced water was added
(20 mL) and the mixture was extracted with ethyl acetate (3
 20 mL). The combined organic phases were dried, filtered and
the solvent was evaporated to give 229 mg (89%) of the desired

1102
R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
4.16. 6-(4-Piperidin-1-ylmethyl-phenyl)-pyrrolo[2,3-
b]pyridine-1-carboxylic acid-4-methoxy-benzylamide (19)
surviving after compound treatment was determined using the
SRB assay. IC50 is defined as the drug concentration producing
50% decrease of cell growth. At least three independent experi-
ments were performed. The resistance index as the ratio between
IC50 of resistant and sensitive cells was evaluated.
To a suspension of 18 (1.700 g, 4.7 mmol) in 3.5:1 DME/H2O
(18 mL) under nitrogen, 1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxa-
borolan-2-yl)-benzyl]-piperidine (95%, 1.67 g, 5.2 mmol), NaHCO3
(1.18 g, 14 mmol), PdCl2(dppf)CH2Cl2 (0.139 g, 0.19 mmol) were
added and the resulting mixture was refluxed for 45 min. in the
dark. After evaporation of the solvent the residue was added with
water/ethyl acetate. The phases were separated. The organic layer
was dried, filtered and evaporated to obtain 3.00 g of crude. Purifi-
cation by flash chromatography (CH2Cl2: MeOH 98:2) gave 1.9 g
(89%) of the title compound; mp 93 °C; 1H NMR (CDCl3) d 10.26
(1H, t, J = 4.4 Hz); 8.01 (1H, d, J = 4.0 Hz); 7.97 (1H, d, J = 8.2 Hz);
7.70–7.60 (3H, m); 7.42 (2H, d, J = 8.5 Hz); 7.30 (2H, d;
J = 8.2 Hz); 6.94 (2H, d, J = 8.5 Hz); 6.54 (1H, d, J = 3.96 Hz); 4.67
(2H, d, J = 4.4 Hz); 3.86 (3H, s); 3.52 (2H, s); 2.53–2.31 (4H, m);
1.72–151 (6H, m). Anal. Calcd for C28H30N4O2: C, 73.98; H, 6.65;
N, 12.33. Found: C, 73.77; H, 6.61; N, 12.52.
4.17.4. PARylation assay
HeLa cells were seeded into 96-well Viewplate black micro-
plates, in culture medium. The plates were incubated for 4/6 h, at
37 °C, under 5% CO2 atmosphere, then the test compounds were
added with serial dilutions, over six points. The plates were incu-
bated for 3 h at 37 °C in 5% CO2, then DNA damage was provoked
by addition of 5
200–500 M). As a negative control, cells untreated with H2O2
were used. The plates were kept at 37 °C for 5 min, and then cells
were fixed by addition of ice-cold MeOH (100 L/well) and kept at
lL of H2O2 solution in H2O (final concentration
l
l
À20 °C for 20 min. Following fixation, cells were repeatedly
washed with PBS and then protein PARylation was detected using
the primary PAR mAb (Alexis ALX-804-220, 1:2000) and the sec-
ondary anti-mouse Alexa Fluor 488 antibody (Molecular probes
A11029, 1:3000). Nuclei were stained with the specific nuclear
4.17. Biological assays
dye Draq5 (Alexis, 5 lM). After 3 h incubation at room temperature
The purity of all tested compounds was assessed by HPLC and
was >95%.
in the dark, cells were extensively washed with PBS and, finally,
fluorescence was read on the high content imaging system ‘Oper-
etta’ (Perkin Elmer). Fluorescence signals related to the residual
amount of PAR polymer on nuclear proteins were measured at
the optimal wavelengths (excitation/emission), and identification
of the nuclei within cells was performed by tracking Draq5. The
percent of PAR-positive cells was calculated at last by measuring
the ratio between the numbers of PAR-positive nuclei over the to-
tal number of Draq5-labeled nuclei. Finally, for the active test com-
pounds, the IC50 values were determined on the basis of the
residual enzyme activity in the presence of increasing concentra-
tion of test compounds.
4.17.1. PARP-1 enzyme assay
Enzymatic assay was carried out by means of a highly sensitive
fluorescent assay (HT-F Homogeneous Inhibition Assay; Trevigen)
according to the manufacturer’s instructions. Briefly, damaged
DNA was mixed with increasing amounts of NAD+ (0–100 nM)
and then incubated, for 30 min, with recombinant human PARP-1
enzyme (expressed as GST fusion protein in Escherichia coli cells),
in the presence or absence of serial dilutions (dose-response curve)
of test compounds. Reactions were terminated by the addition of a
stop solution containing resazurin and, finally, after 30 min of
incubation, the resazurin-dependent fluorescence was quantified
by a multiplate reader (Victor; Perkin Elmer). A NAD standard
curve was generated within each assay. IC50 values for inhibitors
were calculated by means of the ALLFIT software.
4.17.5. In vivo tumor model
Female mice, SCID beige of 22–24 g from Charles River were
used. Mice were housed inside cages of makrolon
(33.2 Â 15 Â 13 cm) (4 mice/cage) with stainless steel cover-feed
and sterilized and dust-free bedding cobs. Nude mice were main-
tained in cages with paper filter covers; food and bedding were
sterilized and water was acidified. Animals were housed under a
light–dark cycle, keeping temperature and humidity constant.
Parameters of the animal rooms were assessed as follows:
22 2 °C temperature, 55 10% relative humidity, about 15–20 fil-
tered air changes/hour and 12 h circadian cycle of artificial light (7
a.m., 7 p.m.). At request, the environmental conditions were mon-
itored and the data were retained in Animal Housing Archives.
Experimental protocols were approved by the Ethic Committee
for Animal Experimentation according to the United Kingdom
Coordinating Committee on Cancer Research Guidelines. MX-1 cell
4.17.2. Cell lines
The pancreatic adenocarcinoma Capan 1, the breast cancer
HCC1937 and the endometrial carcinoma HeLa cell lines were ob-
tained from American Type Culture Collection. The ovarian sensi-
tive and multidrug resistant, respectively A2780 and A2780/Dx
were kindly provided by Istituto Nazionale dei Tumori di Milano.
The breast carcinoma MX-1 cell lines were from CLS (Cells Line Ser-
vice). All tumor cell lines were of human origin and were grown in
RPMI-1640, containing L-glutamine supplemented with 10% heat-
inactivated fetal calf serum (FCS, Life Technologies), except than
the HCC1937 cell line which was cultured in IMDM medium plus
20% foetal calf serum.
lines were inoculated subcutaneously (5 Â 106/200
lL/mouse in
RGF matrigel 50:50). Starting 7 days after tumor injection, mice
(8 mice/group) were treated with in the following experimental
groups: Vehicle (10% DMSO+10% of 2-hydroxypropylbetacyclodex-
trin in PBS), ST7710 100 mg/10 mL/kg ip (q2d/wx2w), AZD-2281
(ST7437) 167 mg/10 mL/kg ip (q2d/wx2w). Body weight record-
ings were carried out through the study and mortality was noted.
To evaluate the antitumor activity of the drugs, tumor diameters
were measured biweekly with a Vernier caliper. The formula TV
(mm3) = [length (mm) Â width (mm)2]/2 was used, where the
width and the length are the shortest and the longest diameters
of each tumor, respectively. When tumors reached a weight of
about 0.5 g, mice were sacrificed by cervical dislocation. Toxicity
of the molecule was determined as: body weight loss percent (%
BWL max) = 100À(mean BWday x/mean BWday 1 Â 100), where
4.17.3. Cell sensitivity studies
Cell sensitivity to drugs was evaluated by growth inhibition as-
says based on cell counting (HCC1937 and Capan 1) or SRB assays
(A2780 and A2780/Dx). HCC1937 and Capan 1 cells were seeded in
duplicates into 6-well plates and exposed to drug 24 h later. The
studied compounds were dissolved in dimethylsulfoxide (DMSO)
and then added to culture medium. DMSO concentration in med-
ium never exceeded 0.25%. After 72 h of drug incubation, cells were
harvested for counting with a cell counter. A2780 and A2780/Dx
cells were grown in a volume of 200 lL at approximately 10% con-
fluency in 96-well multititer plates and were allowed to recover for
an additional 24 h. Tumor cells were treated with either varying
concentrations of drugs or solvent for 72 h. The fraction of cells

R. Cincinelli et al. / Bioorg. Med. Chem. 22 (2014) 1089–1103
3. De Vos, M.; Schreiber, V.; Dantzer, F. Biochem. Pharmacol. 2012, 84, 137.
1103
BWx is the mean BW at the day of maximal loss during the treat-
ment and BW1 is the mean BW on the 1st day of treatment. Lethal
toxicity was also evaluated. Efficacy of the molecule was evaluated
as tumor volume inhibition (TVI%) according to the equation:%
TVI = 100–[(mean tumor weight of treated mice/mean tumor
weight of control group) Â 100]. For statistical comparison, TVs
of experimental groups were compared by Mann–Whitney’s test.
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4.17.6. Molecular modeling
The models were built based on the crystal structure of PARP
catalytic domain in complex with novel inhibitors (PDB code
4HHY).35 After the separation of the coordinates of ligands, cofac-
tors, water molecules and enzyme, polar hydrogens were added
with the GROMACS package36 using the GROMOS 53a6 force
field.37 The structures of the ligands were refined using a system-
atic conformer, search followed by geometry optimization of the
lowest energy structure with NWChem (RMS gradient 0.0010).38
Molecular docking experiments were performed with Autodock
4.0, which uses an empirical scoring function based on the free en-
ergy of binding.39,40 The ligands and the PARP catalytic domain
were further processed using the Autodock Tool Kit (ADT).41
Gasteiger-Marsili charges42 were assigned and solvation parame-
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Structures with less than 1.0 Å root-mean-square deviation (rmsd)
were clustered together and representative model of each cluster
was selected based on the most favourable free energy of binding.
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the current study, we used the combined quantum mechanical
molecular mechanics (QM/MM) approach as implemented in
NWChem.38 The combined quantum mechanical molecular
mechanics (QM/MM) approach provides a simple and effective tool
to study localized molecular transformations in large scale systems
such as those encountered in solution chemistry or enzyme catal-
ysis. In this method an accurate but computationally intensive
quantum mechanical (QM) description is only used for the regions
where electronic structure transformations are occurring (e.g.,
bond making and breaking). The rest of the system, whose chemi-
cal identity remains essentially the same, is treated at the approx-
imate classical molecular mechanics (MM) level. The QM/MM
module in NWChem is built as a top level interface between the
classical MD module and various QM modules, managing initializa-
tion, data transfer, and various high level operations. For the QM/
MM calculations, the PARP-ligand systems resulting from the dock-
ing study were first partitioned into a QM subsystem and an MM
subsystem. The reaction system used a smaller QM subsystem con-
sisting of the ligand and residues within 4.5 Å, whereas the rest of
the system (the MM subsystem) was treated using a modified AM-
BER force field. The boundary problem between the QM and MM
subsystems was treated using the pseudo-bond approach. With
this QM/MM system, an iterative optimization procedure was ap-
plied to the QM/MM system, using 3-21G⁄ QM/MM calculations,
leading to an optimized structure for the reactants. The conver-
gence criterion used was set to obtain an energy gradient of
<10À4, using the twin-range cutoff method for nonbonded interac-
tions, with a long-range cutoff of 14 Å and a short-range cutoff of
8 Å.
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