Synthesis and biological evaluation of new 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles as PI3Kα inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.09.001

A series of novel 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b] indoles was synthesized through a newly developed approach. All these compounds were evaluated against DYRK1A, CDK5 and PI3Kα and showed promising inhibitory activities against PI3Kα with most IC₅₀ values in the micromolar range. Among them, compound 18 was strongly considered as the most interesting compound with an IC₅₀ value of 0.091 μM. This series exhibited also significant anti-proliferative effects in various human cancer cell lines including those resulting in activation of the PI3K pathway.

Full text of DOI:10.1016/j.ejmech.2012.09.001

European Journal of Medicinal Chemistry 57 (2012) 225e233
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new 5-benzylated 4-oxo-3,4-dihydro-5H-
pyridazino[4,5-b]indoles as PI3Ka inhibitors
,
b
c
c
Amélie Bruel a, Cédric Logé a , Marie-Ludivine de Tauzia , Myriam Ravache , Rémy Le Guevel ,
Christiane Guillouzo c, Jean-François Lohier d, Jana Sopkova-de Oliveira Santos e, Olivier Lozach f,
Laurent Meijer f, Sandrine Ruchaud f, Hélène Bénédetti b, Jean-Michel Robert a
*
a Université de Nantes, Nantes Atlantique Universités, Laboratoire de Chimie Thérapeutique, Cibles et Médicaments des Infections et du Cancer, IICIMED-EA 1155,
UFR Sciences Pharmaceutiques, 1 rue Gaston Veil, Nantes F-44035 Cedex 1, France
b Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Université d’Orléans et INSERM, rue Charles Sadron, 45071 Orléans Cedex 2,
France
c Plate-forme ImPACcell, UMS-3480, Université de Rennes 1, Campus de Villejean, 2 avenue du Professeur Léon Bernard, 35043 Rennes Cedex, France
d Laboratoire de Chimie Moleculaire et Thioorganique (LCMT), UMR CNRS 6507, INC3M, FR 3038, Labex EMC3, ENSICAEN & Universite de Caen, 14050 Caen, France
e Centre d’Etude et de Recherche sur le Médicament de Normandie (CERMN), UPRES EA 4258, FR CNRS 3038, INC3M, UFR des Sciences Pharmaceutiques,
Université de Caen Basse-Normandie, Boulevard Becquerel, 14032 Caen Cedex, France
f CNRS-USR3151, Protein Phosphorylation & Human Disease, Station Biologique, B.P.74, F-29682 Roscoff Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2012
Received in revised form
30 August 2012
Accepted 1 September 2012
Available online 10 September 2012
A series of novel 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles was synthesized through
a newly developed approach. All these compounds were evaluated against DYRK1A, CDK5 and PI3K
showed promising inhibitory activities against PI3K with most IC50 values in the micromolar range.
Among them, compound 18 was strongly considered as the most interesting compound with an IC50
value of 0.091 M. This series exhibited also significant anti-proliferative effects in various human cancer
cell lines including those resulting in activation of the PI3K pathway.
Ó 2012 Elsevier Masson SAS. All rights reserved.
a and
a
m
Keywords:
4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]
indoles
Phosphoinositide 3-kinase (PI3K)
Dual-specificity tyrosine phosphorylation-
regulated kinase 1A (DYRK1A)
Kinase inhibitors
Anti-proliferative effects
1. Introduction
of the pyridinyl ring, respectively (PDB code: 3ANR) [2,3]. Due to
the biological importance of these compounds, we aimed to
investigate new representatives of this tricyclic ring system based
on a 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indole scaffold able to
modulate a kinase activity through a competitive inhibition at the
ATP binding site (Fig. 1c). These indole-fused pyridazinones could
contain the molecular features required for a high affinity [4]: key
hydrogen bonds which could interact with the so-called hinge part
present in each kinase but especially various substituted benzyl
peripheral groups attached to the N-5 position of this privileged
structure which could target a pocket not addressed by ATP to gain
binding affinity and selectivity. On the basis of this strategy,
twenty-one compounds were synthesized and evaluated both in
selected kinase assays (the dual-specificity kinase DYRK1A, the
serine/threonine kinase CDK5 and the phosphoinositide 3-kinases
(PI3Ks), which are therapeutic targets in neurodegenerative
During the past few years, the pyridazino[4,5-b]indole scaffold
(Fig.1a), an “aza-carboline”, has found considerable pharmaceutical
interest, and a large number of derivatives have shown a wide
range of biological activities [1]. The natural b-carboline harmine
itself (Fig. 1b), was recently shown to inhibit dual-specificity tyro-
sine phosphorylation-regulated kinase 1A (DYRK1A) activity,
a
kinase implicated in neurodegenerative diseases as Down
syndrome and Alzheimer disease, by interacting with residues
Leu241 (hinge region) and Lys188 in the ATP-binding pocket via
two hydrogen bonds to the methoxy group and the nitrogen atom
* Corresponding author. Tel.: þ33 (0)2 40 41 11 08; fax: þ33 (0)2 40 41 28 76.
E-mail address: cedric.loge@univ-nantes.fr (C. Logé).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.001

226
A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
N
a
b
N
N
N
H
N
H
O
Harmine
pyridazino[4,5-b]indole
"hinge region ?"
c
N
N
R1
NH
NH
O
O
"hinge region ?"
O
N
H
N
R1 = OCH3, F, Br
X = 3 or 4-NO2, CN, OCH3
"affinity pocket ?"
X
Targeted compounds
Fig. 1. Structures of pyridazino[4,5-b]indole (a), harmine (b) and the design concept of 5-benzylated-4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indole.
diseases or cancer) and for their in vitro anti-proliferative potencies
against six human cancer cell lines. As these compounds were
made accessible by short and convenient synthetic pathways, they
could constitute ideal starting points for hit discovery.
The results are summarized in Table 1. All the 5-benzylated series
(15e32) were considered inactive on both DYRK1A and CDK5/p25
since they displayed IC50 values above 10
mM, while they showed
promising inhibitory activities against PI3K
a
with most IC50 values
in the micromolar range. The 8-brominated molecule 18 bearing
a cyano substituent at the meta position of the benzyl group was
strongly considered as the most interesting compound with an IC50
2. Chemistry
value of 0.091 mM, only 10-fold upper than that of PI-103, the well-
5-substituted 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles
are generally prepared by introduction of the desired alkyl residue
at the indole nitrogen before the formation of the pyridazinone
unit, using 2-alkoxycarbonyl-3-acylindole as the substrate [5,6].
Interestingly, Haider et al. have shown that 5-alkyl-4-methyl-2,5-
dihydro-1H-pyridazino[4,5-b]indole-1-ones can be prepared by
selective monoalkylation of N-5 in the parent compound with
stochiometric amounts of alkylating agents [7]. Taking into account
these two main observations, we decided to apply a similar strategy
for the condensed pyridazino[4,5-b]indole-4-one scaffold.
The synthetic route to obtain the 5-benzylated compounds is
shown in Scheme 1. Key intermediates ethyl indole-2-carboxylates
(4, 7, 8) can be prepared by intra-molecular cyclization of the corre-
sponding ethyl pyruvate 4-bromophenylhydrazone (3) in poly-
phosphoric acid (PPA) as a catalyst, according to Fischer’s indole
synthesis or by esterification of commercially 5-fluoro (5) and 5-
methoxyindol-2-carboxylic acids (6). FriedeleCrafts acylation using
aluminium trichloride and acetyl chloride gave indoles 9 and 10 with
good yields. Moderate yield obtained for compounds 11 was due to
the effect of the electron-donating methoxy group which activates
the benzene ring and orients electrophilic attacks in ortho position.
Condensation of hydrazine provided 4-oxo-3,4-dihydro-5H-pyr-
idazino[4,5-b]indoles (12e14). Finally, and as expected, treatment of
these compounds with sodium hydride and 3- or 4-substituted
benzyl bromides afforded the target molecules 15e32. The struc-
ture of 25 was confirmed by a single-crystal X-ray diffraction (Fig. 2).
known dual isoform-selective class I PI3K and mammalian target of
rapamycin (mTOR) inhibitor used as the positive control [8]. This
compound also showed sub-micromolar activity on the p110
gamma isoform of PI3K. These results are not surprising since
tyrosine and serine/threonine kinases are closely related to each
other but only distantly related to the PI3K family [9,10]. Therefore,
as the three-dimensional space in the ATP-binding pocket of PI3K is
larger than the corresponding spaces found in other protein kinases
[11,12], our sterically encumbered 5-benzylated compounds should
preferentially inhibit the lipid kinase family. Interestingly, unsub-
stituted analogues at position 5 (13 and 14) have led to compounds
with micromolar activity on DYRK1A, suggesting that small flat
structures can also be accommodated in the ATP binding site of this
kinase.
3.2. Molecular modeling
To examine possible binding mode for compound 18 inside the
ATP-binding site of PI3K, we have carried out molecular modeling
studies into the existing crystal structure of p110 gamma isoform in
complex with a known inhibitor at 2.40 Å resolution (PDB code,
1E7V) (PDB code: 1E7V) [12] since the co-crystal structure of p110
alpha isoform with a small ligand inhibitor has not been resolved
yet. The docking result indicated that the pyridazinone ring could
form two hydrogen bonding interactions with key PI3K hinge
region residue Val882 (Fig. 3) while the electron-withdrawing
cyano substituent in the meta position of the benzyl ring could
interact with the hydroxyl side chain of Ser806, a residue that is
3. Results and discussion
3.1. Kinase assays
conserved in all PI3Ks (equivalent to Ser774 in PI3Ka) [13]. The
heavier bromine atom is oriented toward a deeper affinity pocket
defined among other by Lys833, Asp841, Ile879 and Asp964, which
is located at the back of the ATP site and was shown to be crucial in
Twenty-one compounds were tested against three representa-
tive kinases: DYRK1A, CDK5/p25 and PI3K p110 isoforms (a and g).

A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
227
O
Br
Br
Br
a
b
O
N
c
NH2
N
H
N
H
NH2
3 (79%)
2 (85%)
1
R1
R1
d
COOEt
COOH
N
N
H
H
5
6
R1 = F
4
7
8
R1 = Br (85%)
R1 = OCH3
R1 = F (95%)
R1 = OCH3 (95%)
O
N
N
R1
R1
R1
NH
O
NH
O
COOEt
e
f
g
R1 = CH3
N
N
H
N
H
12 R1 = Br (79%)
13 R1 = F (72%)
9
10 R1 = F (71%)
R1 = Br (75%)
15-32
11 R1 = OCH3 (51%)
14 R1 = OCH3 (84%)
(44-85%)
X
Scheme 1. Reagents and conditions: (a) (i) NaNO2, HClc, 0 ꢀC, 15 min; (ii) SnCl2, HClc, 0 ꢀC, 4 h; (b) ethyl pyruvate, EtOH, reflux, 5 h; (c) PPA, 85 ꢀC, 3 h; (d) SOCl2, EtOH, reflux, 3 h;
(e) AlCl3, CH3COCl, CH2Cl2, reflux, 3 h or r.t. 5 h; (f) hydrazine hydrate, EtOH, reflux, 5 h; (g) (i) NaH, DMF, r.t., 30 min; (ii) benzyl bromides, DMF, r.t., 8 h.
controlling inhibitor potency [14,15] and could form an additional
halogen bond interaction with the carboxylate side chain of Asp841
(d ¼ 3.085 Å).
All the compounds were found to be as active as PI-103 in all
tumor cell lines showing significant antitumor activity with IC50
values ranging from 0.4 mM for PC3 to 15 mM for HCT-116, but it
clearly appeared that most compounds bearing a fluorine atom at
position C-8 (21, 22 and 26) were the most interesting as they did
not inhibit the growth of normal fibroblasts and they exhibited the
best antitumor activities in the PC3 and MDA-MB-231 cell lines
3.3. Cell effects
Six human cancer cell lines (hepatocellular carcinoma Huh-7,
colorectal adenocarcinoma Caco2, colorectal carcinoma HCT-116,
breast carcinoma MDA-MB-231, prostate carcinoma PC3 and lung
carcinoid NCI-H727) and one normal cell line (fibroblasts) were
used in our experiments. The results are presented in Table 2.
with an IC50 value lower than 1
However, when compared compound 18 which was a potent
PI3K
inhibitor (IC50 ¼ 91 nM, Table 1) and compounds 21, 22 and
26 which were considered inactive on this enzyme (IC50 >10 M),
mM.
a
m
Fig. 2. ORTEP diagram of compound 25.

228
A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
Table 1
Table 2
Kinase inhibition (IC50 values in
highlighted in bold).
mM; NT ¼ not tested ; values lower than 10
m
M are
Anti-proliferative activity in various cell types (IC50 values in
the number in brackets indicate the percentage of cell diminution).
m
M; NT ¼ not tested;
Cpds
Huh7
Caco2 MDA-MB- HCT
PC3
NCI-
H727
Fibroblasts
N
231
116
R1
NH
O
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1.5
1.5
3
1
2
2
2
1.5
2
5
3
3
3
1
1.5
3
1.5
2
4
8
2
3
2
2
2
2
2
2
2
2
2
2
5
6
4
5
4
5
4
4
5
5
5
15
2
4
2
5
4
2
5
4
5
6
2
4
5
4
3
3
7
1.5
5
3
2
5
1
4
3
4
5
5
5
6
4
6
3
2
2.5
4
8
10
20
2
5
2
4
4
N
R1
N
NH
2
O
N
H
2.5
2.5
2
0.6
0.7
1
4
2
3
>25
>25
>25
8
5
>25
4
12-14
X
15-32
0.8
1.5
12
2
0.9
2
.
4
Cpds
R1
X
DYRK1A
CDK5/p25
PI3K
a
PI3Kg
2.5
0.4
3
2.5
(50%)
2 (50%) 2
2
NT
2.5
1.5
12
Br
NT
NT
NT
NT
NT
NT
>10
4.1
>10
13
F
5.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
NT
>10
>10
6
5.1
1.9
0.091
1.5
1.5
>10
>10
>10
5.0
4.2
>10
2.6
14
15
16
17
OMe
Br
Br
5
5
4
2 (50%)
4-NO2
3-NO2
4-CN
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
NT
(50%)
6
3
NT
2
0.9
29
30
31
32
2
2
2
3
NT
3
15
4
5
NT
2
1
5
5
NT
1
Br
4
NT
3
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Br
Br
Br
F
F
F
F
F
3-CN
0.295
5.2
2.2
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.15
NT
NT
1
1.5
(40%)
4-OMe
3-OMe
4-NO2
3-NO2
4-CN
PI-103
3 (30%) 1.5 (30%)
Roscovitine 12
5
12
7
10
20 >25
3-CN
4-OMe
3-OMe
4-NO2
3-NO2
4-CN
3-CN
4-OMe
3-OMe
F
suppressor PTEN (phosphatase with tensin homology)) [16]; the
same observation applies to colorectal HCT-116 carcinoma cell with
a mutation in the p110
suggest that the anti-proliferative effects do not seem to be corre-
lated with an inhibition of the PI3K p110
other mechanisms could be involved.
OMe
OMe
OMe
OMe
OMe
OMe
1.8
2.9
3.3
>10
6.3
a kinase domain [16]. Such results may
a isoform alone and that
PI-103
Roscovitine
0.008
NT
11
0.2
4. Conclusion
we can be surprised to observe that these last three molecules
showed the best antitumor activities in the prostate PC3 carcinoma
cell line which has a well-known genetic abnormality resulting in
activation of the PI3K pathway (e.g. deletion of the tumor
In summary, we reported the original synthesis and preliminary
biological evaluation of new 5-benzylated 4-oxo-3,4-dihydro-5H-
pyridazino[4,5-b]indoles with promising in vitro inhibitory activi-
ties against PI3K
a and significant anti-proliferative effects in
various cell types. However, even if the structureeactivity rela-
tionships can be explained among the three proposed enzymes, the
correlation between the PI3K
a inhibitory activities and some
observed cell effects is not obvious. Further investigations including
downstream mediators of the PI3K pathway (Akt and mTOR
kinases), cell cycle progression and induction of apoptosis should
be done to refine the exact mechanism of these antitumoral agents.
5. Experimental
5.1. Chemistry
Melting points were determined using an Electrothermal
IA9300 digital melting point apparatus and reported uncorrected.
1H and 13C NMR spectra were recorded on a Bruker AC250
(250 MHz) or on Bruker Avance 400 spectrometer (400 MHz).
Chemical shifts are expressed as
d values (ppm) relative to tetra-
methylsilane as internal standard (in NMR description, s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quadruplet, sext ¼ sextuplet,
m ¼ multiplet and b ¼ broad). Coupling constants J are given in
Hertz. IR spectra were obtained in KBr pellets using a PerkineElmer
Paragon FTIR 1000 PC spectrometer. Only the most significant
absorption bands have been reported. Electrospray mass spectro-
metric analysis was performed on a Waters Acquity UPLC System
Fig. 3. Proposed binding mode for compound 18 into the ATP-binding site of PI3K
g
(PDB code: 1E7V). The hydrogens bonds are indicated as yellow dotted lines. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
229
ZQ 2000 single quadrupole. All reactions were monitored by thin-
5.1.5. General procedure for the synthesis of ethyl 3-acetyl-1H-
indole-2-carboxylates (9e11)
layer chromatography (TLC) using 0.2 mm-thick silica gel plates
60F-254 (5735 Merck). Column chromatography was carried out
using silica gel 60 (70e230 Mesh, ASTM, Merck). Chemicals and
solvents used were commercially available.
To a solution of the appropriate ester (4, 7 or 8) (19.30 mmol) in
freshly distilled CH2Cl2 (70 mL) was added aluminium chloride
(38.60 mmol) and dropwise acetyl chloride (57.90 mmol). The
mixture was boiled under argon (compounds 9 and 10) for 3 h or
was stirred at room temperature (compound 11) for 5 h, then
poured into ice-cold water and extracted with CH2Cl2. The organic
layers were combined, dried over Na2SO4, filtered and concentrated
under reduced pressure. The obtained residue was purified over
silica gel chromatography eluting with CH2Cl2.
5.1.1. 4-Bromophenylhydrazine (2)
To an ice-cold aqueous solution of 12 N HCl (15 mL) was added
4-bromoaniline (4.00 g, 23.25 mmol) and dropwise an ice-cold
solution of NaNO2 (1.60 g, 23.19 mmol) in H2O (5 mL). The reac-
tion was stirred for 15 min at 0 ꢀC. To the resulting mixture was
added dropwise an ice-cold solution of tin(II) chloride (13.22 g,
69.73 mmol) in 12 N HCl (40 mL). The reaction mixture was stirred
for 4 h at 0 ꢀC. The precipitate was filtered, washed with 1 N HCl and
poured into water where pH value was adjusted to 10 by adding 1 N
NaOH. The crude product was extracted with Et2O, dried over
Na2SO4, filtered and concentrated under reduced pressure to give
5.1.5.1. Ethyl
3-acetyl-5-bromo-1H-indole-2-carboxylate
(9).
White solid. Yield 75%; 1H NMR (400 MHz, DMSO-d6)
d
12.65 (s, 1H,
NH), 8.12 (d, 1H, J ¼ 1.6 Hz, Harom), 7.49 (d, 1H, J ¼ 8.8 Hz, Harom),
7.46 (dd, 1H, J ¼ 8.8 and 1.6 Hz, Harom), 4.43 (q, 2H, J ¼ 7.2 Hz, CH2),
2.60 (s, 3H, CH3), 1.37 (t, 3H, J ¼ 7.2 Hz, CH3).
an orange solid. Yield 85%; 1H NMR (400 MHz, DMSO-d6)
d 7.25 (d,
2H, J ¼ 8.8 Hz, Harom), 6.97 (s, 1H, NH), 6.76 (d, 2H, J ¼ 8.8 Hz,
5.1.5.2. Ethyl
3-acetyl-5-fluoro-1H-indole-2-carboxylate
(10).
12.62
Harom), 4.40 (s, 2H, NH2).
Off-white solid. Yield 71%; 1H NMR (400 MHz, DMSO-d6)
d
(s, 1H, NH), 7.71 (dd, 1H, J ¼ 9.2 and 2.4 Hz, Harom), 7.57 (dd, 1H,
J ¼ 9.2 and 4.6 Hz, Harom), 7.25 (ddd, 1H, J ¼ 9.2; 9.2 and 2.4 Hz,
Harom), 4.45 (q, 2H, J ¼ 7.2 Hz, CH2), 2.63 (s, 3H, CH3), 1.41 (t, 3H,
J ¼ 7.2 Hz, CH3).
5.1.2. Ethyl pyruvate 4-bromophenylhydrazone (3)
A mixture of 4-bromophenylhydrazine (2) (3.55 g, 18.98 mmol),
ethyl pyruvate (2.65 g, 22.78 mmol) in EtOH (20 mL) was boiled
under argon for 5 h. The cooled reaction mixture was then filtered
and the precipitate was washed with water. The crude product was
triturated with cyclohexane and filtered to give a yellow solid. Yield
5.1.5.3. Ethyl 3-acetyl-5-methoxy-1H-indole-2-carboxylate (11).
White solid. Yield 51%; 1H NMR (400 MHz, DMSO-d6)
d 12.38 (s,
79%; 1H NMR (400 MHz, DMSO-d6)
d
9.97 (s, 1H, NH), 7.48 (d, 2H,
1H, NH), 7.46 (d, 1H, J ¼ 2.4 Hz, Harom), 7.45 (d, 1H, J ¼ 8.8 Hz,
Harom), 7.02 (dd, 1H, J ¼ 8.8 and 2.4 Hz, Harom), 4.40 (q, 2H,
J ¼ 7.0 Hz, CH2), 3.81 (s, 3H, CH3), 2.62 (s, 3H, OCH3), 1.40 (t, 3H,
J ¼ 7.0 Hz, CH3).
J ¼ 8.8 Hz, Harom), 7.25 (d, 2H, J ¼ 8.8 Hz, Harom), 4.23 (q, 2H,
J ¼ 7.2 Hz, CH2), 2.09 (s, 3H, CH3), 1.30 (t, 3H, J ¼ 7.2 Hz, CH3).
5.1.3. Ethyl 5-bromo-1H-indole-2-carboxylate (4)
A mixture of ethyl pyruvate 4-bromophenylhydrazone (3)
(2.23 g, 7.82 mmol) and polyphosphoric acid (22 g) was heated to
85 ꢀC for 3 h. The reaction mixture was then cooled, poured into
ice-cold water and neutralized with saturated aqueous sodium
bicarbonate. The crude product was extracted with EtOAc, dried
over Na2SO4, filtered and concentrated under reduced pressure to
give a pale yellow solid. Yield 85%; 1H NMR (400 MHz, DMSO-d6)
5.1.6. General procedure for the synthesis of 4-oxo-3,4-dihydro-5H-
pyridazino[4,5-b]indoles (12e14)
To a stirred solution of the appropriate ethyl 3-acetyl-1H-
indole-2-carboxylates (9e11) (3.67 mmol) in EtOH (60 mL) was
added dropwise hydrazine hydrate (7.34 mmol). The reaction was
heated under reflux for 6 h, and stirred at room temperature for
16 h. After cooling, the resulting solution was concentrated under
reduced pressure. The residue was washed with 1 N HCl, filtered
and triturated with a small amount of diisopropyl ether.
d
12.10 (s, 1H, NH), 7.87 (d, 1H, J ¼ 0.8 Hz, Harom), 7.42 (dd, 1H,
J ¼ 8.8 and 0.8 Hz, Harom), 7.37 (dd, 1H, J ¼ 8.8 and 1.2 Hz, Harom),
7.12 (d, 1H, J ¼ 1.2 Hz, Harom), 4.34 (q, 2H, J ¼ 7.2 Hz, CH2), 1.34 (t,
3H, J ¼ 7.2 Hz, CH3).
5.1.6.1. 8-bromo-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino[4,5ꢁ-b1]
indole (12). Off-white solid. Yield 79%; mp >300 ꢀC; IR (KBr, cm
)
5.1.4. General procedure for the synthesis of ethyl 1H-indole-2-
carboxylates (7, 8)
3205 (NH), 1655 (CO), 790 (CeBr); 1H NMR (400 MHz, DMSO-d6)
13.01 (s, 1H, NH), 12.68 (s, 1H, NH), 8.23 (d, 1H, J ¼ 1.6 Hz, Harom),
7.66 (dd, 1H, J ¼ 8.8 and 1.6 Hz, Harom), 7.62 (d, 1H, J ¼ 8.8 Hz,
Harom), 2.72 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
155.33,
d
To a stirred solution of the appropriate acid (5 or 6) (11.16 mmol)
in EtOH (60 mL) was added dropwise thionyl chloride (13.40 mmol)
and the mixture was heated under reflux for 3 h. After cooling,
water was added till a precipitate appeared. The crude product was
filtered and dried over reduced pressure.
d
141.88, 137.71, 132.30, 129.49, 124.32, 123.15, 116.57, 115.24, 113.94,
20.10; MS (ESI) m/z (%): 278.0 [M þ H]þ; 280.0 [M þ Hþ2]þ (Br
isotope).
5.1.4.1. Ethyl 5-fluoro-1H-indole-2-carboxylate (7). Off-white solid.
5.1.6.2. 8-fluoro-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino[4,5ꢁ-b1]
)
Yield 95%; 1H NMR (400 MHz, DMSO-d6)
d
11.99 (s, 1H, NH), 7.44
indole (13). Off-white solid. Yield 72%; mp >300 ꢀC; IR (KBr, cm
(dd, 1H, J ¼ 9.2 and 4.8 Hz, Harom), 7.41 (dd, 1H, J ¼ 9.8 and 2.8 Hz,
Harom), 7.13 (ddd, 1H, J ¼ 9.2; 9.2 and 2.8 Hz, Harom), 7.12 (d, 1H,
J ¼ 2.0 Hz, Harom), 4.33 (q, 2H, J ¼ 7.2 Hz, CH2), 1.33 (t, 3H,
J ¼ 7.2 Hz, CH3).
3205 (NH), 1655 (CO); 1H NMR (400 MHz, DMSO-d6)
d 12.90 (s, 1H,
NH), 12.63 (s, 1H, NH), 7.90 (dd, 1H, J ¼ 9.3 and 2.4 Hz, Harom), 7.66
(dd, 1H, J ¼ 9.2 and 4.8 Hz, Harom), 7.40 (ddd, 1H, J ¼ 9.2; 9.2 and
2.4 Hz, Harom), 2.73 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d
157.95 (J ¼ 235.55 Hz), 155.44, 142.05, 135.70, 132.71, 121.66,
5.1.4.2. Ethyl 5-methoxy-1H-indole-2-carboxylate (8). White solid.
117.27, 115.44, 114.57, 107.10, 19.92; MS (ESI) m/z (%): 218.11
[M þ H]þ.
Yield 95%; 1H NMR (400 MHz, DMSO-d6)
d 11.77 (s, 1H, NH), 7.38
(dd, 1H, J ¼ 8.8 and 0.8 Hz, Harom), 7.13 (d, 1H, J ¼ 2.4 Hz, Harom),
7.08 (d, 1H, J ¼ 0.8 Hz, Harom), 6.95 (dd, 1H, J ¼ 8.8 and 2.4 Hz,
Harom), 4.36 (q, 2H, J ¼ 7.1 Hz, CH2), 3.79 (s, 3H, OCH3), 1.37 (t, 3H,
J ¼ 7.1 Hz, CH3).
5.1.6.3. 8-methoxy-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino[4,5-
b]indole (14). Off-white solid. Yield 84%; mp >300 ꢀC; IR (KBr,
cmꢁ1) 3277 (NH), 1643 (CO), 1219, 1047 (CeOeC); 1H NMR

230
A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
(400 MHz, DMSO-d6)
d
12.64 (s, 1H, NH), 12.52 (s, 1H, NH), 7.57 (d,
5.1.7.5. 8-bromo-5-(4-methoxybenzyl)-1-methyl-4-oxo-3,4-dihydro-
5H-pyridazino[4,5-b]indole (19). Off-white solid. Yield 53%; mp
>300 ꢀC; IR (KBr, cmꢁ1) 3480 (NH), 1650 (CO), 1250, 1031 (CeOeC),
1H, J ¼ 8.8 Hz, Harom), 7.52 (d, 1H, J ¼ 2.4 Hz, Harom), 7.21 (dd, 1H,
J ¼ 8.8 and 2.4 Hz, Harom), 3.91 (s, 3H, OCH3), 2.76 (s, 3H, CH3). 13
C
(100.6 MHz, DMSO-d6)
d
155.55, 154.92, 142.05, 134.09, 131.72,
780 (CeBr); 1H NMR (400 MHz, DMSO-d6)
d 12.78 (s, 1H, NH), 8.25
121.93, 117.25, 117.01, 114.09, 103.37, 55.76, 20.19; MS (ESI) m/z (%):
(d,1H, J ¼ 1.6 Hz, Harom), 7.81 (d,1H, J ¼ 8.8 Hz, Harom), 7.69 (d,1H,
J ¼ 8.8 Hz, Harom), 7.26 (d, 2H, J ¼ 8.5 Hz, Harom), 6.86 (d, 2H,
J ¼ 8.5 Hz, Harom), 6.05 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 2.73 (s, 3H,
230.14 [M þ H]þ.
5.1.7. General procedure for the synthesis of 5-benzyl-4-oxo-3,4-
dihydro-5H-pyridazino[4,5-b]indoles (15e32)
CH3). 13C (100.6 MHz, DMSO-d6)
d 158.81, 155.85, 142.07, 137.99,
130.16, 129.72, 129.57, 128.80 (2C), 124.67, 122.61, 116.74, 114.62,
114.29, 114.18 (2C), 55.22, 46.94, 20.22; MS (ESI) m/z (%): 398.0
[M þ H]þ; 400.0 [M þ H þ 2]þ (Br isotope).
To a solution of the appropriate 4-oxo-3,4-dihydro-5H-pyr-
idazino[4,5-b]indoles (12e14) (0.92 mmol) in dry DMF (3 mL) was
added sodium hydride (1.01 mmol, 60% w/w dispersion oil). The
reaction mixture was stirred for 30 min at room temperature. The
benzyl bromide (1.01 mmol) was slowly added dropwise. After
being stirred at room temperature for 16 h, the solution was
concentrated under reduced pressure. The resulting crude product
was washed with water, filtered and triturated with EtOAc. The
desired product was purified over silica gel chromatography eluting
with CH2Cl2 to CH2Cl2/EtOH (98:2).
5.1.7.6. 8-bromo-5-(3-methoxybenzyl)-1-methyl-4-oxo-3,4-dihydro-
5H-pyridazino[4,5-b]indole (20). Off-white solid. Yield 70%; mp
>300 ꢀC; IR (KBr, cmꢁ1) 3451 (NH), 1654 (CO), 1266, 1042 (CeOeC),
779 (CeBr); 1H NMR (400 MHz, DMSO-d6)
d 12.78 (s, 1H, NH), 8.29
(d, 1H, J ¼ 1.6 Hz, Harom), 7.79 (d, 1H, J ¼ 8.8 Hz, Harom), 7.70 (dd,
1H, J ¼ 8.8 and 1.6 Hz, Harom), 7.20 (dd, 1H, J ¼ 8.1 and 7.8 Hz,
Harom), 6.88 (s, 1H, Harom), 6.82 (dd, 1H, J ¼ 8.2 and 2.1 Hz,
Harom), 6.74 (d,1H, J ¼ 7.8 Hz, Harom), 6.09 (s, 2H, CH2), 3.70 (s, 3H,
5.1.7.1. 8-bromo-1-methyl-5-(4-nitrobenzyl)-4-oxo-3,4-dihydro-5H-
OCH3), 2.75 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d 158.81, 155.85,
pyridazino[4,5-b]indole (15). Off-white solid. Yield 78%; mp
142.07, 137.99, 130.16, 129.72, 129.57, 128.80 (2C), 124.67, 122.61,
116.74, 114.62, 114.29, 114.18 (2C), 55.22, 46.94, 20.22; MS (ESI) m/z
(%): 398.0 [M þ H]þ; 400.0 [M þ H þ 2]þ (Br isotope).
>300 ꢀC; IR (KBr, cmꢁ1); 1H NMR (400 MHz, DMSO-d6)
d 12.81 (s,
1H, NH), 8.32 (d, 1H, J ¼ 1.8 Hz, Harom), 8.18 (d, 2H, J ¼ 8.8 Hz,
Harom), 7.78 (d, 1H, J ¼ 9.0 Hz, Harom), 7.72 (dd, 1H, J ¼ 9.0 and
1.8 Hz, Harom), 7.44 (d, 2H, J ¼ 8.8 Hz, Harom), 6.29 (s, 2H, CH2),
5.1.7.7. 8-fluoro-1-methyl-5-(4-nitrobenzyl)-4-oxo-3,4-dihydro-5H-
pyridazino[4,5-b]indole (21). White solid. Yield 67%; mp >300 ꢀC; IR
(KBr, cmꢁ1) >3200 (NH), 1659 (CO),1343 (NO2); 1H NMR (400 MHz,
2.77 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d 155.74, 147.01, 145.27,
142.08, 138.04, 130.25, 130.01, 128.13 (2C), 124.79, 124.03 (2C),
122.61, 117.00, 114.91, 113.89, 47.15, 20.21; MS (ESI) m/z (%): 413.0
[M þ H]þ; 415.0 [M þ H þ 2]þ (Br isotope).
DMSO-d6)
d
12.78 (s,1H, NH), 8.19 (d, 2H, J ¼ 8.8 Hz, Harom), 7.98 (dd,
1H, J ¼ 9.6 and 2.4 Hz, Harom), 7.84 (dd, 1H, J ¼ 9.2 and 4.4 Hz,
Harom), 7.50 (ddd, 1H, J ¼ 9.2; 9.2 and 2.4 Hz, Harom), 7.45 (d, 2H,
J ¼ 8.2 Hz Harom), 6.29 (s, 2H, CH2), 2.75 (s, 3H, CH3).13C (100.6 MHz,
5.1.7.2. 8-bromo-1-methyl-5-(3-nitrobenzyl)-4-oxo-3,4-dihydro-5H-
pyridazino[4,5-b]indole (16). Off-white solid. Yield 44%; mp
>300 ꢀC; IR (KBr, cmꢁ1) 3232 (NH), 1656 (CO), 783 (CeBr); 1H NMR
DMSO-d6)
d
158.39 (J ¼ 236.41 Hz), 155.86, 147.01, 145.42, 142.12,
135.95,130.67,128.17 (2C),124.04 (2C),121.28,117.60,115.96,113.35,
(400 MHz, DMSO-d6)
d
12.85 (s, 1H, NH), 8.32 (d, 1H, J ¼ 1.8 Hz,
107.78, 47.07, 20.09; MS (ESI) m/z (%): 353.04 [M þ H]þ.
Harom), 8.25 (s, 1H, Harom), 8.14 (d, 1H, J ¼ 7.8 Hz, Harom), 7.89 (d,
1H, J ¼ 8.8 Hz, Harom), 7.74 (dd, 1H, J ¼ 8.8 and 1.8 Hz, Harom), 7.66
(d, 1H, J ¼ 7.3 Hz, Harom), 7.62 (dd, 1H, J ¼ 7.3 and 7.8 Hz, Harom),
6.27 (s, 2H, CH2), 2.77 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
5.1.7.8. 8-fluoro-1-methyl-5-(3-nitrobenzyl)-4-oxo-3,4-dihydro-5H-
pyridazino[4,5-b]indole (22). Off-white solid. Yield 67%; mp
>300 ꢀC; IR (KBr, cmꢁ1) 3450 (NH), 1657 (CO), 1340 (NO2); 1H NMR
d
155.80, 148.08, 142.13, 139.82, 137.98, 133.85, 130.49, 130.15,
(400 MHz, DMSO-d6) d 12.81 (s, 1H, NH), 8.27 (s, 1H, Harom), 8.14
130.04, 124.80, 122.75, 122.60, 122.13, 117.03, 114.91, 113.95, 46.76,
20.20; MS (ESI) m/z (%): 413.0 [M þ H]þ; 415.0 [M þ H þ 2]þ (Br
isotope).
(d, 1H, J ¼ 7.6 Hz, Harom), 7.98e7.90 (m, 2H, Harom), 7.47e7.69 (m,
3H, Harom), 6.27 (s, 2H, CH2), 2.74 (s, 3H, CH3). 13C (100.6 MHz,
DMSO-d6)
d 155.92, 148.10, 142.17, 139.97, 135.91, 133.90, 130.57,
130.50, 122.76, 122.71, 121.33, 116.13, 115.86, 113.46, 46.78, 20.10 (2
5.1.7.3. 4-[(8-bromo-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino
[4,5-b]indolyl)methyl] benzonitrile (17). Off-white solid. Yield 52%;
mp >300 ꢀC; IR (KBr, cmꢁ1) 3449 (NH), 2224 (CN), 1659 (CO), 782
carbones not visible); MS (ESI) m/z (%): 353.10 [M þ H]þ.
5.1.7.9. 4-[(8-fluoro-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino
[4,5-b]indolyl)methyl] benzonitrile (23). Off-white solid. Yield 65%;
mp >300 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 2229 (CN), 1659 (CO); 1H
(CeBr); 1H NMR (400 MHz, DMSO-d6)
d 12.81 (s, 1H, NH), 8.32 (d,
1H, J ¼ 1.8 Hz, Harom), 7.79 (d, 2H, J ¼ 8.4 Hz, Harom), 7.77 (d, 1H,
J ¼ 8.8 Hz, Harom), 7.72 (dd, 1H, J ¼ 8.8 and 1.8 Hz, Harom), 7.37 (d,
2H, J ¼ 8.4 Hz, Harom), 6.23 (s, 2H, CH2), 2.77 (s, 3H, CH3). 13C
NMR (400 MHz, DMSO-d6)
d
12.77 (s, 1H, NH), 7.97 (dd, 1H, J ¼ 9.4
and 4.1 Hz, Harom), 7.82 (dd, 1H, J ¼ 9.1 and 4.3 Hz, Harom), 7.80 (d,
2H, J ¼ 8.1 Hz, Harom), 7.48 (ddd, 1H, J ¼ 9.1; 9.1 and 4.1 Hz, Harom),
7.39 (d, 2H, J ¼ 8.1 Hz, Harom), 6.24 (s, 2H, CH2), 2.76 (s, 3H, CH3).
(100.6 MHz, DMSO-d6)
d 155.76, 143.27, 142.11, 138.06, 132.83 (2C),
130.26, 130.01, 127.89 (2C), 124.81, 122.61, 118.78, 116.99, 114.89,
113.93, 110.47, 47.22, 20.22; MS (ESI) m/z (%): 393.0 [M þ H]þ; 395.0
[M þ H þ 2]þ (Br isotope).
13C (100.6 MHz, DMSO-d6)
d
158.19 (J ¼ 238.46 Hz), 155.86, 143.38,
142.09, 135.93, 132.81 (2C), 130.65, 127.90 (2C), 121.28, 118.76,
117.60, 116.01, 113.25, 110.44, 107.78, 47.22, 20.08; MS (ESI) m/z (%):
333.13 [M þ H]þ.
5.1.7.4. 3-[(8-bromo-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino
[4,5-b]indolyl)methyl] benzonitrile (18). Off-white solid. Yield 56%;
mp >300 ꢀC; IR (KBr, cmꢁ1); 1H NMR (400 MHz, DMSO-d6)
d
12.81
5.1.7.10. 3-[(8-fluoro-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino
[4,5-b]indolyl)methyl] benzonitrile (24). Off-white solid. Yield 45%;
mp >300 ꢀC; IR (KBr, cmꢁ1) 3159 (NH), 2233 (CN), 1656 (CO); 1H
(s, 1H, NH), 8.32 (d, 1H, J ¼ 1.6 Hz, Harom), 7.89e7.80 (m, 2H,
Harom), 7.79e7.70 (m, 2H, Harom), 7.58e7.50 (m, 2H, Harom), 6.19
(s, 2H, CH2), 2.76 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d
155.84,
NMR (400 MHz, DMSO-d6)
Harom), 7.71 (m, 1H, Harom), 7.55e7.40 (m, 3H, Harom), 6.14 (s, 2H,
CH2), 2.70 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
158.13
(J ¼ 236.45), 155.71, 141.91, 139.14, 135.65, 131.87, 131.36, 130.75,
d 12.74 (s, 1H, NH), 7.97e7.78 (m, 3H,
142.14, 139.24, 137.98, 132.05, 131.61, 130.93, 130.24, 130.19, 130.00,
124.80, 122.66, 118.76, 117.03, 114.86, 113.96, 111.75, 46.79, 20.22;
MS (ESI) m/z (%): 393.0 [M þ H]þ; 395.0 [M þ H þ 2]þ (Br isotope).
d

A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
231
130.41, 129.96, 121.13, 118.54, 117.37, 115.82, 113.20, 111.51, 107.64,
mp >300 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 2224 (CN), 1661 (CO),
1213, 1041 (CeOeC); 1H NMR (400 MHz, DMSO-d6)
12.68 (s, 1H,
46.58, 19.88; MS (ESI) m/z (%): 333.10 [M þ H]þ.
d
NH), 7.81 (s, 1H, Harom), 7.72e7.78 (m, 2H, Harom), 7.57 (d, 1H,
J ¼ 2.2 Hz, Harom), 7.55e7.49 (m, 2H, Harom), 7.24 (dd, 1H, J ¼ 9.2
and 2.2 Hz, Harom), 6.15 (s, 2H, CH2), 3.90 (s, 3H, OCH3), 2.77 (s, 3H,
5.1.7.11. 8-fluoro-5-(4-methoxybenzyl)-1-methyl-4-oxo-3,4-dihydro-
5H-pyridazino[4,5-b]indole (25). Off-white solid. Yield 70%; mp
>300 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 1654 (CO), 1246, 1020 (CeOe
CH3). 13C (100.6 MHz, DMSO-d6)
d 156.04, 155.45, 142.26, 139.66,
C); 1H NMR (400 MHz, DMSO-d6)
d
12.74 (s, 1H, NH), 7.92 (dd, 1H,
134.32, 132.06, 131.51, 130.90, 130.17, 129.65, 121.53, 118.80, 117.56,
117.34, 112.79, 111.68, 103.99, 55.83, 46.66, 20.31; MS (ESI) m/z (%):
345.10 [M þ H]þ.
J ¼ 9.2 and 2.4 Hz, Harom), 7.87 (dd, 1H, J ¼ 9.2 and 4.4 Hz, Harom),
7.57 (ddd, 1H, J ¼ 9.2; 9.2 and 2.4 Hz, Harom), 7.28 (d, 2H, J ¼ 8.6 Hz,
Harom), 6.86 (d, 2H, J ¼ 8.6 Hz, Harom), 6.05 (s, 2H, CH2), 3.70 (s,
3H, OCH3), 2.73 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d
158.78,
5.1.7.17. 8-methoxy-5-(4-methoxybenzyl)-1-methyl-4-oxo-3,4-
158.22 (J ¼ 236.81 Hz),155.94,142.05,135.88,130.53,129.68,128.84
(2C), 121.20, 117.20, 115.61, 114.15 (2C), 113.65, 107.50, 55.19, 46.93,
20.09; MS (ESI) m/z (%): 338.17 [M þ H]þ.
dihydro-5H-pyridazino[4,5-b]indole (31). Off-white solid. Yield
52%; mp ¼ 247e248 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 1652 (CO),
1
1245, 1030 (CeOeC); H NMR (400 MHz, DMSO-d6)
d 12.63 (s, 1H,
NH), 7.75 (d, 1H, J ¼ 9.2 Hz, Harom), 7.54 (d, 1H, J ¼ 2.4 Hz, Harom),
7.25 (d, 2H, J ¼ 8.7 Hz, Harom), 7.23 (dd, 1H, J ¼ 9.2 and 2.4 Hz,
Harom), 6.85 (d, 2H, J ¼ 8.7 Hz, Harom), 6.02 (s, 2H, CH2), 3.89 (s,
3H, OCH3), 3.70 (s, 3H, OCH3), 2.76 (s, 3H, CH3). 13C (100.6 MHz,
5.1.7.12. 8-fluoro-5-(3-methoxybenzyl)-1-methyl-4-oxo-3,4-
dihydro-5H-pyridazino[4,5-b]indole (26). Off-white solid. Yield
85%; mp >300 ꢀC; IR (KBr, cmꢁ1) >3200 (NH),1654 (CO),1264,1036
(CeOeC); 1H NMR (400 MHz, DMSO-d6)
d
12.75 (s, 1H, NH), 7.98e
DMSO-d6) d 158.71, 156.04, 155.26, 142.14, 134.38, 129.97, 129.57,
7.80 (m, 2H, Harom), 7.47 (m, 1H, Harom), 7.20 (m, 1H, Harom),
128.76 (2C), 121.45, 117.29, 117.02, 114.09 (2C), 113.09, 103.77, 55.79,
6.95e6.70 (m, 3H, Harom), 6.10 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 2.74
55.18, 46.79, 20.29; MS (ESI) m/z (%): 350.14 [M þ H]þ.
(s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d
159.53,158.25 (J ¼ 246.55),
155.89, 142.07, 139.27, 136.01, 130.65, 129.94, 121.08, 119.22, 117.26,
115.50,113.54,113.43,112.57,107.35, 55.15, 47.40, 20.08; MS (ESI) m/
z (%): 338.05 [M þ H]þ.
5.1.7.18. 8-methoxy-5-(3-methoxybenzyl)-1-methyl-4-oxo-3,4-
dihydro-5H-pyridazino[4,5-b]indole (32). Off-white solid. Yield
52%; mp ¼ 247e248 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 1655 (CO),
1267, 1038 (CeOeC); 1H NMR (400 MHz, DMSO-d6)
d 12.64 (s, 1H,
5.1.7.13. 8-methoxy-1-methyl-5-(4-nitrobenzyl)-4-oxo-3,4-dihydro-
5H-pyridazino[4,5-b]indole (27). Off-white solid. Yield 64%;
mp ¼ 288e289 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 1659 (CO), 1342
NH), 7.70 (d, 1H, J ¼ 9.2 Hz, Harom), 7.53 (d, 1H, J ¼ 2.2 Hz, Harom),
7.23 (dd, 1H, J ¼ 9.2 and 2.2 Hz, Harom), 7.19 (dd, 1H, J ¼ 8.0 and
7.6 Hz, Harom), 6.86 (s, 1H, Harom), 6.81 (dd, 1H, J ¼ 8.0 and
2.4 Hz, Harom), 6.74 (d, 1H, J ¼ 7.6 Hz, Harom), 6.07 (s, 2H, CH2),
3.90 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 2.77 (s, 3H, CH3). 13C
(NO2), 1215, 1035 (CeOeC); 1H NMR (400 MHz, DMSO-d6)
d 12.68
(s, 1H, NH), 8.18 (d, 2H, J ¼ 8.8 Hz, Harom), 7.69 (d, 1H, J ¼ 9.0 Hz,
Harom), 7.57 (d, 1H, J ¼ 2.4 Hz, Harom), 7.43 (d, 2H, J ¼ 8.8 Hz,
Harom), 7.24 (dd, 1H, J ¼ 9.0 and 2.4 Hz, Harom), 6.27 (s, 2H, CH2),
3.91 (s, 3H, OCH3), 2.79 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
(100.6 MHz, DMSO-d6)
d 159.51, 156.02, 155.30, 142.17, 139.58,
134.48, 129.90, 129.67, 121.41, 119.20, 117.36, 117.06, 113.38, 112.99,
112.43, 103.76, 55.78, 55.12, 47.27, 20.29; MS (ESI) m/z (%): 350.14
[M þ H]þ.
d
155.97, 155.48, 146.96, 145.75, 142.22, 134.38, 129.69, 128.12 (2C),
124.03 (2C), 121.51, 117.58, 117.34, 112.73, 103.96, 55.83, 46.94,
20.31; MS (ESI) m/z (%): 365.13 [M þ H]þ.
5.2. Biological evaluation
5.1.7.14. 8-methoxy-1-methyl-5-(3-nitrobenzyl)-4-oxo-3,4-dihydro-
5H-pyridazino[4,5-b]indole (28). Off-white solid. Yield 64%;
mp ¼ 288e289 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 1662 (CO), 1348
5.2.1. Kinase assays on CDK5/p25 and DYRK1A
Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM TriseHCl
(pH 7.5), 50 mg heparin/mL. Buffer C: 60 mM b-glycerophosphate,
(NO2), 1213, 1040 (CeOeC); 1H NMR (400 MHz, DMSO-d6)
d
12.70
15 mM p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 5 mM
EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 1 mM
phenylphosphate. Kinase activities were assayed in Buffer A or C, at
(s, 1H, NH), 8.21 (s, 1H, Harom), 8.12 (d, 1H, J ¼ 7.2 Hz, Harom), 7.79
(d, 1H, J ¼ 9.2 Hz, Harom), 7.65 (d, 1H, J ¼ 7.6 Hz, Harom), 7.63 (dd,
1H, J ¼ 7.6 and 7.2 Hz, Harom), 7.56 (d, 1H, J ¼ 2.2 Hz, Harom), 7.25
(dd, 1H, J ¼ 9.2 and 2.2 Hz, Harom), 6.24 (s, 2H, CH2), 3.90 (s, 3H,
30 ꢀC, at a final ATP concentration of 15
mM. Blank values were
subtracted and activities expressed in % of the maximal activity, i.e.
in the absence of inhibitors. Controls were performed with
appropriate dilutions of DMSO. CDK5/p25 (human, recombinant)
was prepared as previously described [17]. Its kinase activity was
assayed in buffer C, with 1 mg histone H1/mL, in the presence of
OCH3), 2.77 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d 156.06,155.53,
148.13, 142.38, 140.31, 134.38, 133.90, 130.50, 129.63, 122.72, 122.06,
121.55, 117.67, 117.42, 112.80, 101.01, 55.86, 46.71, 20.33; MS (ESI) m/
z (%): 365.20 [M þ H]þ.
15
30
m
m
M [
g
-33P] ATP (3000 Ci/mmol; 10 mCi/mL) in a final volume of
5.1.7.15. 4-[(8-methoxy-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino
[4,5-b]indolyl)methyl] benzonitrile (29). Off-white solid. Yield 56%;
mp >300 ꢀC; IR (KBr, cmꢁ1) >3200 (NH), 2216 (CN), 1660 (CO),
L. After 30 min incubation at 30 ꢀC, 25
m
L aliquots of super-
natant were spotted onto 2.5 ꢂ 3 cm pieces of Whatman P81
phosphocellulose paper, and, 20 s later, the filters were washed five
times (for at least 5 min each time) in a solution of 10 mL phos-
phoric acid/L of water. The wet filters were counted in the presence
1210, 1041 (CeOeC); 1H NMR (400 MHz, DMSO-d6)
d 12.68 (s, 1H,
NH), 7.80 (d, 2H, J ¼ 8.4 Hz, Harom), 7.69 (d, 1H, J ¼ 9.2 Hz, Harom),
7.57 (d, 1H, J ¼ 2.4 Hz, Harom), 7.36 (d, 2H, J ¼ 8.4 Hz, Harom), 7.23
(dd, 1H, J ¼ 9.2 and 2.4 Hz, Harom), 6.20 (s, 2H, CH2), 3.90 (s, 3H,
of
1 mL ACS (Amersham) scintillation fluid. DYRK1A (rat,
recombinant, expressed in Escherichia coli as a GST fusion protein)
was purified by affinity chromatography on glutathione-agarose
and assayed as described for CDK5/p25 using myelin basic
protein (1 mg/mL) as a substrate.
OCH3), 2.78 (s, 3H, CH3). 13C (100.6 MHz, DMSO-d6)
d 155.98, 155.47,
143.72, 142.24, 134.40, 132.80 (2C), 129.70, 127.89 (2C), 121.49,
118.83, 117.58, 117.32, 112.76, 110.36, 103.96, 55.83, 47.11, 20.31; MS
(ESI) m/z (%): 345.10 [M þ H]þ.
5.2.2. Kinase assays on PI3K (alpha and gamma)
5.1.7.16. 3-[(8-methoxy-1-methyl-4-oxo-3,4-dihydro-5H-pyridazino
[4,5-b]indolyl)methyl] benzonitrile (30). Off-white solid. Yield 52%;
The activity of the alpha isoform of PI3k was evaluated using
a purified heterodimer (ref PV4788 from Invitrogen) composed of

232
A. Bruel et al. / European Journal of Medicinal Chemistry 57 (2012) 225e233
the catalytic p110
a
subunit (PIK3CA) and the regulatory p85
a
5.3. Crystallographic analysis
subunit (PIK3R1). The activity of the gamma isoform was evaluated
using the purified catalytic p110 subunit (PIK3CG) (ref PV4786
g
Single crystals of compound 25 suitable for X-ray crystallo-
graphic analysis were obtained by slow diffusion from compound
25 solution (dichloromethane/diisopropyl ether). X-ray diffraction
experiment was performed with graphiteemonochromatized Mo
from Invitrogen). The ADAPTAÔ kinase assay kit (Invitrogen) is
a fluorescence-based immunoassay for the detection of ADP. It
takes place in 384-well plates and can be divided into two phases:
a kinase reaction phase, and an ADP detection phase. In the kinase
reaction phase, the following components are mixed into the same
Ka radiation on
a BrukereNonius Kappa CCD area detector
diffractometer at 296 K. The data treatment was carried out with
APEX II program [18]. The crystal structure was solved by direct
methods using SHELXTL package [19] and refined by SHELX97 [20].
All non-hydrogen atoms were refined anisotropically and the
hydrogen atoms were located from the electron density map and
refined isotropically. Crystallographic data for compound 25 have
been deposited at the Cambridge Crystallographic Data Centre,
CCDC ID 889555. Copies of this information may be obtained free of
charge from the Director, CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK (þ44-1223-336408; E-mail: deposit@ccdc.cam.ac.ukor
http://www.ccdc.cam.ac.uk).
well: 5
diluted in kinase assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl,
0.03% CHAPS, 1 mM EGTA, 3 mM MgCl2, 2 mM DTT), 2.5 L of
kinase at the optimal concentration (222 ng/mL) diluted in kinase
assay buffer, and 2.5 L of serial dilutions of inhibitors diluted in 4%
mL of a solution containing 20 mM ATP and 2 mM PIP2
m
m
DMSO and kinase assay buffer. For each inhibitor concentration,
the assay was run in triplicate. The reaction was then incubated
60 min at room temperature. For the ADP detection phase, 5 mL of
a detection solution containing Europium-labeled anti-ADP anti-
body (6 nM), Alexa FluorÒ 647 labeled ADP tracer (12 nM), and
EDTA (30 mM, to stop the kinase reaction), diluted in TR-FRET
dilution buffer (provided by the manufacturer) is added to the
assay well. After 30 min of incubation at room temperature, the
384-well plates were read in a Victor V plate reader configured for
HTRF (Perkin Elmer). Excitation was performed at 340 nm and
emission was measured at 665 nm and 615 nm. The inhibition
curve was then plotted as emission ratio 665 nm/615 nm versus
inhibitor concentration. In order to calculate substrate conversion
values (% conversion of ATP in ADP) from the raw TReFRET
emission ratios, an ATPeADP titration curve was performed with
5.4. Molecular modeling
Molecular modeling studies were performed using Sybyl soft-
ware version 8.0 [21] running on a dell precision T3400 worksta-
tion. The three-dimensional structure of compound 18 was built
from a standard fragments library, and its geometry was subse-
quently optimized using the Tripos force field [22] including the
electrostatic term calculated from Gasteiger and Hückel atomic
charges. Powell’s method available in Maximin2 procedure was
used for energy minimization until the gradient value was smaller
a total nucleotide concentration of 10 mM. The resulting TReFRET
emission ratio was plotted against the % conversion of ATP to
ADP. The data of this curve were fit to a 3-parameter hyperbolic
model with the following equation obtained from a GraphPadÔ
PrismÒ software:
than 0.001 kcal/mol Å. The structure of PI3K
g in complex with
inhibitor LY294002 (PDB code, 1E7V) [12] was used as the template
for docking. The original ligand as well as the water molecules were
removed from the coordinates set. Flexible docking of molecule 18
into the ATP-binding site was performed using GOLD software [23].
The most stable docking model was selected according to the best
scored conformation predicted by the GoldScore scoring function.
Y ¼ C þ A ꢂ ð1 ꢁ ðX=ðB þ XÞÞÞ:
The % conversion corresponding to each TReFRET ratio was
calculated using the following equation:
Acknowledgments
ðC þ A ꢁ RatioÞ
% Conversion ¼ B ꢂ
ðRatio ꢁ CÞ
This work was supported by grant from the Canceropôle Grand
Ouest.
The % conversion was then plotted versus the concentration of
inhibitor. The amount of inhibitor required to elicit a 50% change in
the % conversion of ATP in ADP (the EC50) was then determined.
This EC50 corresponds to the IC50 value of the inhibitor.
Appendix A. Supplementary material
1H and 13C NMR spectra for compounds 12, 13, 18 and 25 can be
found, in the online version at http://dx.doi.org/10.1016/j.ejmech.
2012.09.001.
5.2.3. Cell culture and survival assay
Skin diploid fibroblastic cells were provided by BIOPREDIC
International Company (Rennes, France). Caco2 (REF ECACC:
86010202), Huh-7D12 (REF ECACC: 01042712), MDA-MB-231 (REF
ECACC: 92020424), HCT-116 (REF ECACC: 91091005), PC3 (REF
ECACC: 90112714) and NCI-H727 (REF ECACC: 94060303) cell lines
were obtained from the ECACC collection. Cells were grown
according to ECACC recommendations. The toxicity test of the
compounds on these cells was as follows: 2 ꢂ 103 cells for HCT-116
cells or 4 ꢂ 103 for the other cells were seeded in 96 multiwell
plates in triplicate and left for 24 h for attachment, spreading and
growing. Then, cells were exposed for 48 h to increasing concen-
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